Edited by Hu DK
Published online Apr 15, 2002. doi: 10.3748/wjg.v8.i2.282
Revised: January 7, 2002
Accepted: January 15, 2002
Published online: April 15, 2002
AIM: To set up a new method to detect tissue inhibitors of metalloproteinase-1 and -2 (TIMP-1 and TIMP-2) in sera of patients with hepatic cirrhosis, and to investigate the expression and location of TIMP-1 and TIMP-2 in liver tissue of patients with hepatic cirrhosis, and the correlation between TIMPs in liver and those in sera so as to discuss whether TIMPs can be used as a diagnosis index of hepatic fibrosis.
METHODS: The monoclonal antibodies (McAbs) of TIMP-1 and TIMP-2 were used to sensitize erythrocytes, and solid-phase absorption to sensitized erythrocytes (SPASE) was used to detect TIMP-1 and TIMP-2 in the sera of patients with hepatic cirrhosis. Meanwhile, with the method of in situ hybridization and immunohistochemistry, we studied the mRNA expression and antigen location of TIMP-1 and TIMP-2 in the livers of 40 hepatic cirrhosis patients with pathologic diagnosis.
RESULTS: With SPASE, they were 16.4% higher in the acute hepatitis group, 33.3% higher in the chronic hepatitis group, and the positive rates were 73.6% and 61.2% respectively in sera of hepatic cirrhosis patients, which were remarkably higher than those in chronic hepatitis and acute hepatitis group (P < 0.001). In 40 samples of hepatic cirrhosis tissues, all of them showed positive expression of TIMP-1 and TIMP-2 mRNA detected with immunohistochemistry or in situ hybridization (positive rate was 100%). Expression of TIMPs in different degrees could be found in liver tissue with cirrhosis. TIMPs were located in cytoplasm of liver cells of patients with hepatic cirrhosis. There was a significant correlation between serum TIMPs level and liver TIMPs level.
CONCLUSION: SPASE is a useful method to detect the TIMP-1 and TIMP-2 in sera of patients with hepatic cirrhosis, and TIMP-1 and TIMP-2 can be considered as a useful diagnostic index of hepatic fibrosis, especially TIMP-1.
- Citation: Nie QH, Cheng YQ, Xie YM, Zhou YX, Bai XG, Cao YZ. Methodologic research on TIMP-1, TIMP-2 detection as a new diagnostic index for hepatic fibrosis and its significance. World J Gastroenterol 2002; 8(2): 282-287
- URL: https://www.wjgnet.com/1007-9327/full/v8/i2/282.htm
- DOI: https://dx.doi.org/10.3748/wjg.v8.i2.282
SPASE (solid-phase absorption to sensitized erythrocytes) is an immunological detecting method possessing the similar principle with ELISA (enzyme-linked immunosorbent assay) and SPRIA (solid-phase radioimmunoassay). Erythrocytes sensitized by antibodies are used as the indicator, taking the place of enzyme or isotope labeled antibody. The result is judged by the hemagglutination phenomena. SPASE has the same sensitivity and specificity as ELISA and SPRIA, and is so simple and rapid as RPHA (reverse passive hemagglutination)[2,3]. The monoclonal antibodies (McAbs) of TIMP-1 and TIMP-2 were used to sensitize erythrocytes. SPASE was used to detect TIMP-1 and TIMP-2 in the sera of patients with hepatic cirrhosis, and proved pretty well. At the same time, with the method of in situ hybridization and immunohistochemistry, the mRNA expression and antigen location of TIMP-1 and TIMP-2 in the livers of 40 hepatic cirrhosis patients diagnosed by pathology were studied.
The U shape 96 well plexiglass microhemagglutination plate was used as the solid phase support. The solid-phase antibody (McAbs of TIMP-1 and TIMP-2) was purchased from Maxim Biologic Technology Corp, America, (No: MAB-0282, MAB-0283).Formaldehyde-chicken or sheep erythrocytes were sensitized by TIMP-1 and TIMP-2 McAb, according to the method introduced by Han to prepare for sensitized erythrocytes. The concentration of McAbs was 50-100 mg•L⁻¹. The positive samples that simulated positive sample were obtained from the sera of hepatic cirrhotic patients pathologiclly diagnosed, and normal human sera were used as the negative control, and PBS as the blank control. To test the thermal stability, McAb was bathed at 60 °Cwater with different time, and its activity was detected by RIA.
We collected 408 serum samples from Tangdu Hospital and Xijing Hospital affiliated to the Fourth Military Medical University, and Southwest Hospital affiliated to the Third Military Medical University, The First and Fourth Hospitals of Chinese PLA. The diagnosis of viral hepatitis accorded with the diagnosis standard revised by the Fifth National Academic Conference for Infectious and Parasitic Diseases. According to the standard, there were 128 serum samples of acute hepatitis, 174 of chronic hepatitis, and 106 of hepatic cirrhosis. 40 liver samples were collected from the pathologic departments of Tangdu and Xijing Hospitals affiliated to the Fourth Military Medical University. All the 40 samples were proved pathologiclly with nodular hepatic cirrhosis. Male, 40; female, 8; the mean age, 50 years.
Procedure of SPASE 50 mg•L⁻¹ McAb were added into U shape 96 well plexiglass micro hemagglutination plate (100 μL•well), to make one layer and the unnecessary solution should be removed, the sparing solution could be reused, and then dried under room temperature, fixed at 56 °C, washed with PBS 3-4 times, to get rid of the unfixed McAb molecule. 100 μL per well of the prepared serum sample (diluted at the ratio volume of 1:10) was added, reacted at 37 °C for 1 h, washed with PBS 3-4 times, and then 50 μL per well of McAb sensitized erythrocytes were added. After shaking mixed, they were standing for 1 h at 37 °C or 2 h at room temperature. The results were determined according to the absorbed condition of erythrocytes (like RPHA). The erythrocytes depositing on the bottom of the well was considered as negative result, showing a spot or a little circle in the center of the well without scattered erythrocytes around. The positive result was determined when the erythrocytes were absorbed and a layer spread on the bottom of the well.
Immunohistochemical staining The laboratory procedure referred to references 6-12.SP immunostaining was performed as described by streptomycin avidin-peroxidase immunochemistry kit (purchased from Maxim Biological Technology Company). Briefly, the liver samples were embedded with paraffin, and serial sections at 4 μm thickness were prepared. Paraffin was removed from the sections with xylene and rehydrated with graded ethanol. After the antigens were repaired, unspecific immunoglobulin-binding sites were blocked by a 20 min preincubation with 100 mL•L⁻¹ normal human sera. The sections were then incubated with monoclonal antibody against TIMP-1 or TIMP-2 at 4 °C overnight, and then secondary antibody was added at 37 °C for 30-40 min, avidin-peroxidase at 37 °C for 20 min, and finally DAB was added to be stained. After the sections were washed several times, they were counterstained with hematoxylin, dehydrated with ethanol, rinsed in xylene, and the sections were mounted with gum for microscopic examination and photography. To make sure of the reliability and specificity of the result of immunohistochemical staining, rabbit sera and PBS were used to replace the first antibody in our control test. 10 normal liver tissues were also used as the normal control samples.
Liver tissue in situ hybridization The investigation procedure referred to related references[13-17]. The in situ hybridization kit was purchased from Boshide Biological Technology Limited Company (Wuhan, China, No. MK1549). In situ hybidization was performed according to the manufacturer's direction. Briefly, the paraffin embedded serial sections (thickness 4 μm), were dried at 80 °C, and their paraffin was removed by xylene and rehydrated with graded ethanol. The sections were acidified in HCl for 30 min, and blocked in 3 mL 300 mL•L⁻¹ H2O2 for 10 min before digestion in proteinase K for 30 min, and then dehydrated with graded ethanol. After prehybridization at 37-40 °C for 2 h, the labeled cDNA probes of TIMP-1 and TIMP-2 were denatured in hybidization buffer at 95 °C for 10 min, then -20 °C for 10 min, added on tissues which had been prehybridized at 37 °C overnight. Sections were washed in turn with 2 × SSC, 1 × SSC, 0.2 × SSC, and Buffer I, blocking water was added at room temperature for 20 min, and then rabbit anti-digoxin serum at 37 °C for 60 min, biotinylated goat anti-rabbit serum at 37 °C for 30 min, SABC at 37 °C for 30 min, finally DAB was added to be stained. After several times of washing, the sections were counterstained with hematoxylin, dehydrated with ethanol, rinsed in xylene, and mounted with gum for microscopic examination and photography. (1) Blank control: prehybridization solution was replaced by the cDNA probes of TIMP-1 and TIMP-2 to be hybridized with the positive liver sections; (2) Negative control: the in situ hybridization was performed with 10 normal liver sections.
Semi-quantitative index was used to determine the results of immunohistochemistry and in situ hybridization: no positive cells (-); positive cells occupied hepatocytes of hepatic lobulfe less than than1/3 (+); 1/3-2/3 (++); more than 2/3 (+++).
The best laboratory condition and influence factors To study the related factor of sensitivity and specificity of this technique, the simulated positive samples were used, and we concluded that there were two main factors might directly influence the experiment condition. One was the concentration of coated antibodies and the other the density of sensitized erythrocytes. Much higher or less concentration of coated antibodies would decrease the sensitivity, while 50-100 mg•L⁻¹ might be the best (Table 1). There was a close relation between the density of sensitized erythrocytes, which is the indicator, and at 2.5•L⁻¹ the sensitivity appeared to be the best (Table 2).
|Coated McAb (mg•L⁻¹)||Serum titer||Negative control|
|Density of sensitized erythrocytes||Serum titer||Negative control|
Degree of accuracy and repetitive test The coefficient of variation (CV) in one lot of samples in the same plate was 6.06%. The coefficient of variation (CV) in different lot of samples in different plate was 7.65%. Both of them were less than 10% (Table 3), which showed that this technique was provided with good degree of accuracy and repetition.
|Well numbers||(¯x ± s)||CV (%)|
|In the same lot||15||0.642 ± 0.0389||6.06|
|In different lot||5||0.575 ± 0.044||7.65|
Serum samples The 408 serum samples of patients with liver disease were used to detect TIMP-1 and TIMP-2 with SPASE. The positive rates were 73.6% and 61.2% respectively, which were remarkablly higher than those in chronic hepatitis and acute hepatitis group. There was a significant statistical difference (P < 0.001, Table 4).
|Positive number||%||Positive number||%|
Immunohistochemistry By immunohistochemistry detection, the positive signal as brown particles were scattered or diffused only in cytoplasm other than nuclei in liver cells. Table 5 represented the result of detecting TIMP-1 and TIMP-2 of 40 liver samples of hepatic cirrhosis, in which, for TIMP-1, 28 samples were (+++), 70.0% of cytoplasma; 4 were (++), 10.0%; 8 were (+), accounted for 20.0%. For TIMP-2, 22 samples were (+++), 55.0% of cytoplasma; 10 were (++), 25.0%; 8 were (+), 20.0%. 10 normal liver tissues were negative. There was no positive signal after abridging the first antibody or the second one, and there was no positive signal when the first antibody replaced by rabbit serum or PBS, which proved that the results of immunohistochemistry detecting were specific.
In situ hybridization The positive signal of in situ hybridization showed brown particles, and distributed in the cytoplasm, scattered or diffused. There was no positive signal in nuclei. All the 40 samples of hepatic cirrhosis tissues showed positive expression of TIMP-1 and TIMP-2 mRNA, and the positive rate was 100%. Table 6 showed the intensity of TIMP-1 and TIMP-2 mRNA expression in the liver samples. For TIMP-1 mRNA, 32 samples were (+++), 80.0% of cytoplasma; 6 were (++), 15.0%; 2 were (+), 5.0%. Of TIMP-2 mRNA, 22 samples were (+++), in which 55.0% of cytoplasma; 16 were (++), accounted for 40.0%; 2 were (+), 5.0%. The expression intensity of TIMP-1 mRNA was stronger than that of TIMP-2 mRNA. There was no positive signal when TIMP-1 cDNA probes or TIMP-2 cDNA probes were replaced by the prehybridization solution, and when the 10 normal liver tissues were hybridized with TIMP-1 cDNA probes or TIMP-2 cDNA probes, all were negative. These proved that the results of in situ hybridization were specific.
SPASE is a new method used to detect the TIMP-1 and TIMP-2 in sera of patient with hepatic cirrhosis. During the setting up of this technique, we preliminarily tried to find out how to treat the influence factors and choose the condition. We consider that this method can not only possess both the advantages of SPA and IHT (indirect hemagglutination test), but also avoid the radiation pollution of RIA or cancer-causing danger of ELISA. Meanwhile, this method possesses many other advantages, such as wider range of usage, less influence factors, saving McAb, being easy to operate and judge detecting results. In a word, this is a specific, sensitive, rapid and economic method. There are two main factors that may influence the laboratory condition. Firstly, lower concentration of coated antibodies may result in fewer antibodies combined on the surface of support, which won't be able to catch antigens, while higher density may result in overlap of antibodies on the surface of support, which will have effect on the space position of conjunction of antigens and antibodies, and then antigens can't be effectively caught. Secondly, acting as the indicator system, a reverse correlation exists between the density of sensitized erythrocyte and sensitivity of detection. When the density of sensitized erythrocyte is higher than 3•L⁻¹, the detection sensitivity will be significantly decreased, while clarity of the result would be impaired if the density is lower than 1•L⁻¹. Because the erythrocytes can only react with one layer of the agent on the surface of solid support, excessive erythrocytes didn't combine with agents and would deposit at the bottom of wells, then the judgment of the results would be affected. For this reason, a layer of the sensitized erythrocytes with adequate density is better. The density we used in practice is about 2.5•L⁻¹, which is lower than that used in RPHA. In present study, the way of coating the antibodies was improved, and proved that the method of heating-fixed antibodies was feasible. Sheep erythrocytes and chicken erythrocytes sensitized by formaldehyde were detected with sensitization test, and no specific difference was observed except a little faster sedimentation rate of the former. This insured the source of erythrocytes.
The occurrence and progress of hepatic cirrhosis were the result of the interaction between hepatocytes and extracellular matrixes (ECM)[18-25]. The increase of ECM synthesis and decrease of ECM degradation will result in excess deposition of ECM in liver. More important reason of the excess deposition of ECM is the decrease of ECM degradation in the late stage. The metrical metalloproteinases played a leading role during the degradation of ECM[26-35]. MMPs were a group of zinc-ion dependent enzymes, which created conditions for further degradation of other proteinases though reducing the stability of helical structure of collagen and changing the secondary structure of substrates. TIMPs were a group of polypeptides with the ability of inhibiting the function of MMPs. TIMPs would inhibit the degradation of ECM through two main ways, which is non-covalent modification or conjugated with proenzyme. Research work showed that TIMP could be divided into four classes: TIMP-1, TIMP-2, TIMP-3, and TIMP-4. However, only TIMP-1 and TIMP-2 could be detected in liver[37-44]. All the 40 samples of hepatic cirrhosis tissues, showed positive expression of TIMP-1 and TIMP-2, and the positive rate was 100%. TIMP-1 and TIMP-2 were expressed at the same time, and were located in the liver cytoplasm, not in the nuclei. The expression of TIMP-1 was more obvious than that of TIMP-2. No expression of related antigens of TIMP-1 and TIMP-2 were detected. All these indicated that TIMP-1 did play an important role in the development of liver fibrosis and liver cirrhosis. The inhibitory effect of MMPs was enhanced with the high level expressing of TIMP-1 and TIMP-2 which resulted in the decrease of degradation of ECM, the deposit of ECM and the development of liver fibrosis and liver cirrhosis. The cause of the higher expression of TIMP-1 probably lay in two main reasons. First, the different classes of MMP resulted in the different inhibition activity of TIMP to MMP. TIMP-1 to procollagenase (MMP1) and TIMP-2 to gelatinase -A (MMP2), and gelatinase -B (MMP9) had assumed stronger inhibition activity. During the degradation of ECM, the main one was MMP1, and collagens I, III were the main object of MMP1. Second, the promotion of TIMP-1 was 10 times more than TIMP-2 to the proliferation of cells (including fibroblast, epithelial cells, endothelial cells, and smooth muscle cells). Furthermore, this action had no relation with the inhibition action of TIMP to MMP[45-59].
In situ hybridization was mainly used to observe the characteristics and accurate location of gene expression. The high sensitivity and strong specificity of this technique will be preferable to study the pathogenesis of hepatic fibrosis and to demonstrate the diagnosis. Recently, digoxin has become a widely used non-isotope labeled compound characterized by its perfect specific and stability. Its sensitivity is almost the same as isotope, but without pollution. So, it is easily accepted and used to label TIMP-1 and TIMP-2 cDNA probe to detect the paraffin sections of liver. The results showed that this technique was high sensitive and specific. The rates of positive expressions of TIMP-1 and TIMP-2 were 80.0% and 55.0% respectively. The location of TIMPs expression was in cytoplasm of hepatocyte, except nuclei, and the mRNA expression of TIMP-1 was stronger than that of TIMP-2, which was in accordance with the results of immunohistochemistry, and further proved that the TIMPs played a key role in the development of hepatic fibrosis and hepatic cirrhosis.
It is now known that there is a noticeable increase of TIMP-1 in the injured liver, which takes place earlier and increases faster, therefore, more and more researchers has regarded it as the diagnostic index of hepatic fibrosis. Detecting TIMP-1 and TIMP-2 with SPASE was used as a quick labortory diagnosis of hepatic fibrohosis. The sera from hepatic cirrhosis patients pathologically confirmed and normal people served as positive and negative control respectively. The results of detecting TIMP-1 and TIMP-2 in the sera of 408 patients with hepatic disease showed that positive rate of TIMPs was higher in sera of hepatic cirrhosis patients than that of acute or chronic hepatitis (P < 0.001). TIMPs in sera of chronic hepatitis patients were apparently higher than those of acute hepatitis patients. This conclusion enumerated above was Supported by the detecting results of TIMP-1 and TIMP-2 by using immunohistochemistry and in situ hybridization.
Recently, Murawaki et al[60-62] has detected TIMP-1 and TIMP-2 in sera of patients with chronic liver disease by means of ELISA, and found a good relation between TIMP-1 and TIMP-2 in sera and in liver. The sensitivity and specificity of TIMP-1 were higher than those of TIMP-2. In injured liver, especially in fibrotic liver, TIMP-1 predominated, and the degree of TIMP-1 was remarkably related to the severity of hepatic fibrosis. Compared with TIMP-1, the specificity and sensitivity of TIMP-2 were inadvisable for diagnosis of hepatic fibrosis. So, TIMP-1 was more important than TIMP-2 in the determination of histological change of hepatic fibrosis[63-70]. This was proved by our study of the expression of related antigens and the location of mRNA of TIMP-1 and TIMP-2. The expression of related antigens in liver could be reflected through detecting TIMPs in sera. So, TIMP-1 and TIMP-2 could be considered as useful diagnostic index of hepatic fibrosis, especially TIMP-1. Because viral hepatitis is common in China[71-99], liver fibrosis is the focus of research[18-24,100,101]. So serum TIMP-1 and TIMP-2 will find wide use in practice in the future.
I acknowledge the advice and help of Prof. Bo-Rong Pan!
|1.||Nie QH, Huang C, Zhang KR, Peng BM. SPASE for rapid detection of Shigella flexneri form fecal samples with monoclonal antibody. Zhonghua Chuanranbing Zazhi. 1995;13:145-148.|
|2.||Nie QH, Huang C, Zhang KR, Peng BM. Preparation of prevalent monoclonal antibodies of Shigella flexneri and their application in rapid diagnosis of shigellosis. Seventh International Congress on Rapid Methods and Automation in Microbiology and immunology (LONDON). Programme Abstracts. 1993;41.|
|3.||Li CW. The modern immunochemistric technique. First ban. Shanghai: Shanghai Sci. & Tech. Publishing House 1992; 235-239.|
|4.||Han CY. Indirect hemagglutinative Technique. Beijing: Sci Publi House 1979; 94-111.|
|5.||The prevention and treatment program of viral hepatitis. Zhonghua Neike Zazhi. 1995;34:788-791.|
|6.||Nie QH, Xie Q, Hu DR, Li MD, Li L. The expression of hepatitis G virus-related antigens in the liver tissue of patients with HGV/GBV-C infection. Disan Junyi Daxue Xuebao. 1997;19:394-396.|
|7.||Nie QH, Hu DR, Li MD, Xie Q. The expression of HGV/GBV-C or HCV related antigens in the liver tissue of patients coinfected with hepatitis C and G viruses. Shijie Huaren Xiaohua Zazhi. 2000;8:114-115.|
|8.||Nie QH, Li MD, Hu DR, Li L. The expression of hepatitis G virus-related antigens in the liver tissues of with hepatitis patients. Zhonghua Chuanranbing Zazhi. 2000;18:173-175.|
|9.||Wang D, Shi JQ, Liu FX. Immunohistochemical detection of proliferating cell nuclear antigen in hepatocellular carcinoma. China Natl J New Gastroenterol. 1997;3:101-103.|
|10.||Zhang LF, Peng WW, Yao JL, Tang YH. Immunohistochemical detection of HCV infection in patients with hepatocellular carcinoma and other liver diseases. World J Gastroenterol. 1998;4:64-65. [PubMed]|
|11.||Yan JP, Liu JC, Ma XH, Jia JB, Zhao YC, Xu RL, Li CM, Han DW. Immunohistochemical study on basic fibroblast growth factor in experimental liver fibrosis. Xin Xiaohuabingxue Zazhi. 1997;5:642-644.|
|12.||Nie QH, Li L, Li MD, Hu DR. Clinical and immunopathological study on GB virus B (GBV-B) infection. Shijie Huaren Xiaohua Zazhi. 2000;8:775-781.|
|13.||Nie QH, Li MD, Hu DR. Detection of hepatitis G virus RNA in liver tissue using digoxigenin labelled probe by in situ hybridization. J Gastroenterol Hepatol. 1999;14:A365.|
|14.||Liu YJ, Cong WM, Xie TP, Wang H, Shen F, Guo YJ, Chen H, Wu MC. Detecting the localization of hepatitis B and C virus in hepatocellular carcinoma by double in situ hybridization. China Natl J New Gastroenterol. 1996;2:187-189.|
|15.||Zhao GQ, Xue L, Xu HY, Tang XM, Hu RD, Dong J. In situ hybridization assay of androgen receptor gene in hepatocarcinogenesis. World J Gastroenterol. 1998;4:503-505. [PubMed]|
|16.||Qian QJ, Xue HB, Qu ZQ, Fang SG, Cao HF, Wu MC. In situ detection of tumor infiltrating lymphocytes expressing perforin and fas ligand genes in human HCC. World J Gastroenterol. 1999;5:12-14. [PubMed]|
|17.||Nie QH, Hu DR, Li MD, Li L, Zhu YH. Detection of hepatitis G virus RNA in liver tissue using digoxigenin labelled probe by in situ hybridization. Shijie Huaren Xiaohua Zazhi. 2000;8:771-774.|
|18.||Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med. 1993;328:1828-1835. [PubMed] [DOI]|
|19.||George DK, Ramm GA, Walker NI, Powell LW, Crawford DH. Elevated serum type IV collagen: A sensitive indicator of the presence of cirrhosis in haemochromatosis. J Hepatol. 1999;31:47-52. [PubMed] [DOI]|
|20.||Kossakowska AE, Edwards DR, Lee SS, Urbanski LS, Stabbler AL, Zhang CL, Phillips BW, Zhang Y, Urbanski SJ. Altered balance between matrix metalloproteinases and their inhibitors in experimental biliary fibrosis. Am J Pathol. 1998;153:1895-1902. [PubMed] [DOI]|
|21.||Arthur MJ, Mann DA, Iredale JP. Tissue inhibitors of metalloproteinases, hepatic stellate cells and liver fibrosis. J Gastroenterol Hepatol. 1998;13 Suppl:S33-S38. [PubMed]|
|22.||Arthur MJ. Role of Ito cells in the degradation of matrix in liver. J Gastroenterol Hepatol. 1995;10 Suppl 1:S57-S62. [PubMed] [DOI]|
|23.||Arthur MJ. Collagenases and liver fibrosis. J Hepatol. 1995;22:43-48. [PubMed]|
|24.||Arthur MJ. Degradation of matrix proteins in liver fibrosis. Pathol Res Pract. 1994;190:825-833. [PubMed] [DOI]|
|25.||Liu XS, Li DG, Lu HM, Xu QF. Effects of tetrandrine and verapamil on fibroblastic growth and proliferation. China Natl J New Gastroenterol. 1997;3:70.|
|26.||Wang YF, Li QF, Wang H, Mao Q, Wu CQ. Effects of vitamin E on experimental hepatic fibrosis in rats. World J Gastroenterol. 1998;4:157.|
|27.||Huang ZG, Zhai WR, Zhang YE, Zhang XR. Study of heteroserum-induced rat liver fibrosis model and its mechanism. World J Gastroenterol. 1998;4:206-209. [PubMed]|
|28.||Jia JB, Han DW, Xu RL, Gao F, Zhao LF, Zhao YC, Yan JP, Ma XH. Effect of endotoxin on fibronectin synthesis of rat primary cultured hepatocytes. World J Gastroenterol. 1998;4:329-331. [PubMed]|
|29.||Cheng ML, Wu YY, Huang KF, Luo TY, Ding YS, Lu YY, Liu RC, Wu J. Clinical study on the treatment of liver fibrosis due to hepatitis B by IFN-alpha (1) and traditional medicine preparation. World J Gastroenterol. 1999;5:267-269. [PubMed]|
|30.||Du WD, Zhang YE, Zhai WR, Zhou XM. Dynamic changes of type I, III and IV collagen synthesis and distribution of collagen-producing cells in carbon tetrachloride-induced rat liver fibrosis. World J Gastroenterol. 1999;5:397-403. [PubMed]|
|31.||Zhu YH, Hu DR, Nie QH, Liu GD, Tan ZX. Study on activation and cfos, cjun expression of in vitro cultured human hepatic stellate cells. Shijie Huaren Xiaohua Zazhi. 2000;8:299-302.|
|32.||Sun ZQ, Wang YJ, Quan QZ, Liu XF, Pan X, Jiang XL. Prevention and treatment action of tetrandrine on experimental liver fibrosis in rats. Xin Xiaohuabingxue Zazhi. 1994;2:19-20.|
|33.||Sun ZQ, Wang YJ, Quan QZ, Han GY, Jin XH. Change of serum phosphonate esterase in hepatic fibrosis in rats. Xin Xiaohuabingxue Zazhi. 1994;2:206-207.|
|34.||Wang YJ, Sun ZQ, Quan QZ, Zhang ZJ. Controlled study of Cordyceps sinensis and colchicine on the antifibrosis effect of liver. Xin Xiaohuabingxue Zazhi. 1994;2:208-209.|
|35.||Quan QZ, Sun ZQ, Li DG, Wang YJ, Che JT, Qi F, Fan P. Experimental and clinical study of calcium channel blockers on antifibrosis in chronic liver diseases. Xin Xiaohuabingxue Zazhi. 1994;2:214-216.|
|36.||Olaso E, Friedman SL. Molecular regulation of hepatic fibrogenesis. J Hepatol. 1998;29:836-847. [PubMed] [DOI]|
|37.||Pinzani M, Marra F, Carloni V. Signal transduction in hepatic stellate cells. Liver. 1998;18:2-13. [PubMed] [DOI]|
|38.||Alcolado R, Arthur MJ, Iredale JP. Pathogenesis of liver fibrosis. Clin Sci (. Lond). 1997;92:103-112. [PubMed] [DOI]|
|39.||Iredale JP. Tissue inhibitors of metalloproteinases in liver fibrosis. Int J Biochem Cell Biol. 1997;29:43-54. [PubMed] [DOI]|
|40.||Yang SM, Fang DC, Feng HF, Chen GM, Gao LQ. Diagnostic value of serum prolidase, procollagen type III and hyaluronic acid levels for liver fibrosis. Xin Xiaohuabingxue Zazhi. 1995;3:27-28.|
|41.||Torres L, García-Trevijano ER, Rodríguez JA, Carretero MV, Bustos M, Fernández E, Eguinoa E, Mato JM, Avila MA. Induction of TIMP-1 expression in rat hepatic stellate cells and hepatocytes: A new role for homocysteine in liver fibrosis. Biochim Biophys Acta. 1999;1455:12-22. [PubMed] [DOI]|
|42.||George DK, Ramm GA, Walker NI, Powell LW, Crawford DH. Elevated serum type IV collagen: A sensitive indicator of the presence of cirrhosis in haemochromatosis. J Hepatol. 1999;31:47-52. [PubMed] [DOI]|
|43.||Arthur MJ, Iredale JP, Mann DA. Tissue inhibitors of metalloproteinases: role in liver fibrosis and alcoholic liver disease. Alcohol Clin Exp Res. 1999;23:940-943. [PubMed] [DOI]|
|44.||Li DG, Lu HM, Chen YW. Studies on anti liver fibrosis of tetrandrine. Shijie Huaren Xiaohua Zazhi. 1999;7:171-172.|
|45.||Sakaida I, Uchida K, Hironaka K, Okita K. Prolyl 4-hydroxylase inhibitor (HOE 077) prevents TIMP-1 gene expression in rat liver fibrosis. J Gastroenterol. 1999;34:376-377. [PubMed] [DOI]|
|46.||Wang FS, Wu ZZ. Current situation in studies of gene therapy for liver cirrhosis and liver fibrosis. Shijie Huaren Xiaohua Zazhi. 2000;8:371-373.|
|47.||Han GY, Jin XH, Wu CQ, Wang HF, Liu WJ. Alteration of serum phosphonate esterase in liver fibrosis in rats. Xin Xiaohuabingxue Zazhi. 1996;4:369-370.|
|48.||Chen F, Chai WM. The progress of diagnosis and therapy for liver fibrosis during the latest three years. Xin Xiaohuabingxue Zazhi. 1997;5:69-71.|
|49.||Yang YX, Kang JY. Mechanism and serum diagnosis of liver fibrosis. Xin Xiaohuabingxue Zazhi. 1997;5:119-120.|
|50.||Hou ZJ, Li HZ, Liu XW. Enzymology in diagnosis of liver fibrosis. Xin Xiaohuabingxue Zazhi. 1997;5:325-326.|
|51.||Qiu DK, Li H, Zeng MD, Li JQ. Effect of cordyceps polysaccharides-liposome on the expression of interstitial mRNA in rats with hepatic fibrosis. Xin Xiaohuabingxue Zazhi. 1997;5:417-418.|
|52.||Yan JP, Liu JC, Ma XH, Jia JB, Zhao YC, Xu RL, Li CM, Han DW. Immunohistochemical study on basic fibroblast growth factor in experimental liver fibrosis. Xin Xiaohuabingxue Zazhi. 1997;5:642-644.|
|53.||Wang X, Chen YX, Xu CF, Zhao GN, Huang YX, Wang QL. Relationship between tumor necrosis factor-α and liver fibrosis. Huaren Xiaohua Zazhi. 1998;6:106-108.|
|54.||Xu LX, Xie XC, Jin R, Ji ZH, Wu ZZ, Wang ZS. Effect of selenium in rat experimental liver fibrosis. Huaren Xiaohua Zazhi. 1998;6:133-135.|
|55.||Wu YA, Kong XT. Anti-hepatic fibrosis effect of pentoxifylling. Shijie Huaren Xiaohua Zazhi. 1999;7:265-266.|
|56.||Wang GQ, Kong XT. Action of cell factor and Decorin in tissue fibrosis. Shijie Huaren Xiaohua Zazhi. 2000;8:458-460.|
|57.||Liu F, Wang XM, Liu JX, Wei MX. Relationship between serum TGFβ1 of chronic hepatitis B and hepatic tissue pathology and hepatic fibrosis quantity. Shijie Huaren Xiaohua Zazhi. 2000;8:528-531.|
|58.||Liu F, Liu JX, Cao ZC, Li BS, Zhao CY, Kong L, Zhen Z. Relationship between TGF β1, serum indexes of liver fibrosis and hepatic tissue pathology in patients with chronic liver diseases. Shijie Huaren Xiaohua Zazhi. 1999;7:519-521.|
|59.||Gu SW, Luo KX, Zhang L, Wu AH, He HT, Weng JY. Relationship between ductule proliferation and liver fibrosis of chronic liver disease. Shijie Huaren Xiaohua Zazhi. 1999;7:845-847.|
|60.||Murawaki Y, Ikuta Y, Idobe Y, Kawasaki H. Serum matrix metalloproteinase-1 in patients with chronic viral hepatitis. J Gastroenterol Hepatol. 1999;14:138-145. [PubMed] [DOI]|
|61.||Murawaki Y, Ikuta Y, Kawasaki H. Clinical usefulness of serum tissue inhibitor of metalloproteinases (TIMP)-2 assay in patients with chronic liver disease in comparison with serum TIMP-1. Clin Chim Acta. 1999;281:109-120. [PubMed] [DOI]|
|62.||Murawaki Y, Ikuta Y, Idobe Y, Kitamura Y, Kawasaki H. Tissue inhibitor of metalloproteinase-1 in the liver of patients with chronic liver disease. J Hepatol. 1997;26:1213-1219. [PubMed] [DOI]|
|63.||Li BS, Wang J, Zhen YJ, Liu JX, Wei MX, Sun SQ, Wang SQ. Experimental study on serum fibrosis markers and liver tissue pathology and hepatic fibrosis in immunodamaged rats. Shijie Huaren Xiaohua Zazhi. 1999;7:1031-1034.|
|64.||Wang Y, Gao Y, Huang YQ, Yu JL, Fang SG. Gelatinase A proenzyme expression in the process of experimental liver fibrosis. Shijie Huaren Xiaohua Zazhi. 2000;8:165-167.|
|65.||Yao XX. Diagnosis and treatment of liver fibrosis. Shijie Huaren Xiaohua Zazhi. 2000;8:681-689.|
|66.||Iredale JP, Murphy G, Hembry RM, Friedman SL, Arthur MJ. Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1. Implications for regulation of matrix degradation in liver. J Clin Invest. 1992;90:282-287. [PubMed] [DOI]|
|67.||Roeb E, Purucker E, Breuer B, Nguyen H, Heinrich PC, Rose-John S, Matern S. TIMP expression in toxic and cholestatic liver injury in rat. J Hepatol. 1997;27:535-544. [PubMed] [DOI]|
|68.||Herbst H, Wege T, Milani S, Pellegrini G, Orzechowski HD, Bechstein WO, Neuhaus P, Gressner AM, Schuppan D. Tissue inhibitor of metalloproteinase-1 and -2 RNA expression in rat and human liver fibrosis. Am J Pathol. 1997;150:1647-1659. [PubMed]|
|69.||Kasahara A, Hayashi N, Mochizuki K, Oshita M, Katayama K, Kato M, Masuzawa M, Yoshihara H, Naito M, Miyamoto T. Circulating matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 as serum markers of fibrosis in patients with chronic hepatitis C. Relationship to interferon response. J Hepatol. 1997;26:574-583. [PubMed] [DOI]|
|70.||Kossakowska AE, Edwards DR, Lee SS, Urbanski LS, Stabbler AL, Zhang CL, Phillips BW, Zhang Y, Urbanski SJ. Altered balance between matrix metalloproteinases and their inhibitors in experimental biliary fibrosis. Am J Pathol. 1998;153:1895-1902. [PubMed] [DOI]|
|71.||Song CH, Wu MY, Wang XL, Dong Q, Tang RH, Fan XL. Correlation between HDV infection and HBV serum markers. China Natl J New Gastroenterol. 1996;2:230-231.|
|72.||Gao JE, Tao QM, Guo JP, Ji HP, Lang ZW, Ji Y, Feng BF. Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins. China Natl J New Gastroenterol. 1997;3:114-116.|
|73.||Yang DH, Xiu C, Yang B, Gu JR, Qian LF, Qu SM. Expression of insulin-like growth factor II and its receptor in liver cells of chronic liver diseases. China Natl J New Gastroenterol. 1997;3:117-118.|
|74.||Yu SJ. A comparative study on proliferating activity between HBV related and HCV related small HCC. China Natl J New Gastroenterol. 1997;3:236-237.|
|75.||Zhang SL, Han XB, Yue YF. Relationship between HBV viremia level of pregnant women and intrauterine infection: neated PCR for detection of HBV DNA. World J Gastroenterol. 1998;4:61-63. [PubMed]|
|76.||Gao FG, Sun WS, Cao YL, Zhang LN, Song J, Li HF, Yan SK. HBx-DNA probe preparation and its application in study of hepatocarcinogenesis. World J Gastroenterol. 1998;4:320-322. [PubMed]|
|77.||Wang WL, Gu GY, Hu M. Expression and significance of HBV genes and their antigens in human primary intrahepatic cholangiocarcinoma. World J Gastroenterol. 1998;4:392-396. [PubMed]|
|78.||Zhoug S, Wen SM, Zhang DF, Wang QL, Wang SQ, Ren H. Sequencing of PCR amplified HBV DNA pre-c and c regions in the 2.2.15 cells and antiviral action by targeted antisense oligonucleotide directed against sequence. World J Gastroenterol. 1998;4:434-436. [PubMed]|
|79.||Zhoug S, Wen SM, Zhang DF, Wang QL, Wang SQ, Ren H. Sequencing of PCR amplified HBV DNA pre-c and c regions in the 2.2.15 cells and antiviral action by targeted antisense oligonucleotide directed against sequence. World J Gastroenterol. 1998;4:434-436. [PubMed]|
|80.||Zheng Z, Yang SW, Xiao W, Sun P, Li XJ, Hu YQ. Evaluation of the HBV in HBsAg by HBV DNA measured with PCR. World J Gastroenterol. 1998;4:S77.|
|81.||Lee JH, Ku JL, Park YJ, Lee KU, Kim WH, Park JG. Establishment and characterization of four human hepatocellular carcinoma cell lines containing hepatitis B virus DNA. World J Gastroenterol. 1999;5:289-295. [PubMed]|
|82.||Guo SP, Ma ZS, Wang WL. Construction of eukaryotic expression vector of HBV x gene. World J Gastroenterol. 1999;5:351-352. [PubMed]|
|83.||Tang RX, Gao FG, Zeng LY, Wang YW, Wang YL. Detection of HBV DNA and its existence status in liver tissues and peripheral blood lymphocytes from chronic hepatitis B patients. World J Gastroenterol. 1999;5:359-361. [PubMed]|
|84.||Wen SJ, Xiang KJ, Huang ZH, Zhou R, Qi XZ. Construction of HBV-specific ribozyme and its recombinant with HDV and their cleavage activity in vitro. World J Gastroenterol. 2000;6:377-380. [PubMed]|
|85.||Wang Y, Liu H, Zhou Q, Li X. Analysis of point mutation in site 1896 of HBV precore and its detection in the tissues and serum of HCC patients. World J Gastroenterol. 2000;6:395-397. [PubMed]|
|86.||Hu YP, Yao YC, Li JX, Wang XM, Li H, Wang ZH, Lei ZH. The cloning of 3'-truncated preS/S gene from HBV genomic DNA and its expression in transgenic mice. World J Gastroenterol. 2000;6:734-737. [PubMed]|
|87.||Wei L, Wang Y, Chen HS, Tao QM. Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing. China Natl J New Gastroenterol. 1997;3:12-15.|
|88.||Zhou P, Cai Q, Chen YC, Zhang MS, Guan J, Li XJ. Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C. China Natl J New Gastroenterol. 1997;3:108-110.|
|89.||Sun DG, Liu CY, Meng ZD, Sun YD, Wang SC, Yang YQ, Liang ZL, Zhuang H. A prospective study of vertical transmission of hepatitis C virus. China Natl J New Gastroenterol. 1997;3:111-113.|
|90.||Gao JE, Tao QM, Guo JP, Ji HP, Lang ZW, Ji Y, Feng BF. preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins. China Natl J New Gastroenterol. 1997;3:114-116.|
|91.||Zhang LF, Peng WW, Yao JL, Tang YH. Immunohistochemical detection of HCV infection in patients with hepatocellular carcinoma and other liver diseases. World J Gastroenterol. 1998;4:64-65. [PubMed]|
|92.||Zhu FL, Lu HY, Li Z, Qi ZT. Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E. coli. World J Gastroenterol. 1998;4:165-168. [PubMed]|
|93.||Soresi M, Carroccio A, Bonfissuto G, Agate V, Magliarisi C, Aragona F, Levrero M, Notarbartolo A, Montalto G. Ultrasound detection of abdominal lymphadenomegaly in subjects with hepatitis C virus infection and persistently normal transaminases: A predictive index of liver histology severity. J Hepatol. 1998;28:544-549. [PubMed] [DOI]|
|94.||Yang JM, Wang RQ, Bu BG, Zhou ZC, Fang DC, Luo YH. Effect of HCV infection on expression of several cancer-associated gene products in HCC. World J Gastroenterol. 1999;5:25-27. [PubMed]|
|95.||Feng DY, Chen RX, Peng Y, Zheng H, Yan YH. Effect of HCV NS (3) protein on p53 protein expression in hep atocarcinogenesis. World J Gastroenterol. 1999;5:45-46. [PubMed]|
|96.||Huang F, Zhao GZ, Li Y. HCV genotypes in hepatitis C patients and their clinical significances. World J Gastroenterol. 1999;5:547-549. [PubMed]|
|97.||Dai YM, Shou ZP, Ni CR, Wang NJ, Zhang SP. Localization of HCV RNA and capsid protein in human hepatocellular carcinoma. World J Gastroenterol. 2000;6:136-137. [PubMed]|
|98.||Dai YM, Shou ZP, Ni CR, Wang NJ, Zhang SP. Localization of HCV RNA and capsid protein in human hepatocellular carcinoma. World J Gastroenterol. 2000;6:136-137. [PubMed]|
|99.||Cheng JL, Liu BL, Zhang Y, Tong WB, Yan Z, Feng BF. Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction. World J Gastroenterol. 2001;7:370-375. [PubMed]|
|100.||Nie QH, Li MD, Hu DR, Chen GZ. Study on the cause of human protective immunodeficiency after HCV infection. Shijie Huaren Xiaohua Zazhi. 2000;8:28-30.|
|101.||Nie QH, Cheng YQ, Xie YM, Zhou YX, Cao YZ. Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis. World J Gastroenterol. 2001;7:363-369. [PubMed]|