| P.O.Box 2345, Beijing 100023,China | China Nati J New Gastroenterol 1996 Dec 2;(4):212-214 |
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Study
on mechanism of treating acute hepatic failure with cryopreserved porcine
hepatocyte xenotransplantation
Cong
Lin, Kuan Yong Hou, Wen Xiu Zhang,
Jing Ning Lu, Xiao Si Zhou,Yan Dao Gong,
Xiao Lang Ding, Xiu Fang Zhang, Nan Ming Zhao
Subject
headings liver failure, acute; cryopreservation, liver/cytology;
transplantation, heterologous; membrane proteins
Lin C,Hou KY,Zhang WX,Lu JN,Zhou XS,Gong YD,Ding XL,Zhang XF,Zhao NM. Study on
mechanism of treating acute hepatic failure with cryopreserved porcine
hepatocyte xenotransplantation.China Nati J New Gastroenterol,1996;2(4):212-214
Absrtact
AIM To study the effect of
hypothermia on structure of membrane proteins of pig hepatocytes and the
mechanism of treating acute hepatic failure in Wistar rats with transplantation
of cryopreserved porcine hepatocytes.
METHODS Isolated
porcine hepatocytes were cryopreserved in liquid nitrogen for 150 days and then
transplanted to the rat abdominal cavity. Three days later, 80% of rat liver by
weight was resected. Conformation of membrane proteins of the porcine
hepatocytes, liver function and weight of the remaining liver in the rats were
measured.
RESULTS Viability of
the cryopreserved hepatocytes was 78%. Light and electron microscopy
showed that the hepatocytes were morphologically intact. The cells were
rich in glycogen and glucose-6-phosphatase. |á-helix
content in the membrane
proteins of fresh cells was 48%. After freezing the cells, the content was
increased to 59.5%. Survival rate of rats in the transplanted group was 16/17,
significantly higher than 3/9 in the control group (P=0.0022). However, there
were no significant differences in the weight of the remaining liver and indices
of liver function between the two groups.
CONCLUSION Transplant
of frozen porcine hepatocytes was effective in treating
INTRODUCTION
Hepatocyte transplantation has been applied to the treatment of acute hepatic failure
(AHF) and congenital disturbed hepatic diseases［1］.
It was shown that with cryopreservation of hepatocytes for long-term,
the shape and functions of
hepatocytes could be maintained［2］ and
AHF could be treated effectively［3］. The
immunogenicity of tissues and cells decreased obviously after freeze［4］, but
the mechanism is not known completely. In our experiment,
we explored the effect of hypothermia on antigen structure of pig hepatocellular
membranes and the mechanism of treating AHF in Wistar rats with transplantation
of the cryopreserved porcine hepatocytes.
MATERIALS AND METHODS
Preparation of pig hepatocytes and histologic examination
A Chinese experimental minipig［5］
weighing 7kg was used as the donor. After gradient cooling, the isolated porcine
hepatocytes［6］ were
preserved in liquid nitrogen (-196℃)
for 150 days［3］. Before
transplantation, the cell viability was measured and the hepatocytes were
observed under light and electron
microscope. HE staining and histochemical staining for glycogen were used in
light microscopic observations. The percentage of cells containing glucose-6-phosphatase
(G6Pase) ++-++++
was
counted. Fresh and cryopreserved hepatocytes of the pig were cultured routinely
for 48 hours.
Preparation of hepatocellular membrane proteins and conformational
measurement
Porcine hepatocytes were washed with 0.05
M boric acid, 0.15M
NaCl, 1mM MgCl2, 1mM CaCl2 and 0.1mM
phenylmethylsulfonyl fluoride (PMSF), pH 7.2.
The cells were centrifuged at 450×g for 5min and then lysed
with 100 volumes of 0.02M boric acid, 0.2mM
EDTA and 0.1mM
PMSF, pH 10.2.
Whole cells were removed by centrifugation at 12000×g for 30min. Cell
membranes were then suspended in PBS, pH 7.4,
layered on 35% sucrose w/v in PBS and centrifuged at 7000×g
for 30min. The material at the sucrose-PBS interface was membrane proteins［7］.
Their conformations, were calculated from their circular dichroism spectra.
Animal groups and pig hepatocyte transplantation
Wistar rats weighing 200-250kg were used, and 80% of liver by weight was resected
to induce AHF. The AHF rats usually died within 7 days after the operation［3］. The
experimental animals were divided into two parts. The
first part was used to observe the survival rate and their living situation
during the survival for one month after 80% hepatectomy. This part
was further divided into two groups: transplant group (Ⅰt, 17 rats) and control group (Ⅰc,
9 rats). The
second part was sacrificed on the 2nd or 7th day following AHF to measure the
liver function and the weight of the remaining liver in the AHF rats, and to do
histological examination. This part was divided into two groups too: transplanted
group (Ⅱt,
37 rats) and control group (Ⅱc, 27
rats). Fifteen normal rats provided comparative standards of the liver weight
and function. 4×107
cryopreserved hepatocytes in
2ml were injected into the abdominal cavity of the rats in the two
transplanted groups before and after 80% hepatectomy［6］.
Normal saline was injected into the rats of the two control groups.
Biochemical values of liver function
Biochemical values of liver function included serum concentrations of albumin (ALB),
total bilirubin (TBr) and direct bilirubin (DBr).
Statistical analyses
Data were processed with variance analysis and q
test.
RESULTS
The viability of fresh pig hepatocytes was 84%, and that of cryopreserved cells
was 78%. Light microscopy showed that the frozen hepatocytes had the shape of
sphere and were rich in glycogen and G6Pase. The hepatocytes containing G6Pase ++-++++
accounted
for 97% of total liver cells. There was no apparent difference between the
cryopreserved cells and fresh ones under electron microscope. The
cells grew very well in culture and there was no difference either.
As regards to the
conformation of membrane proteins, the α helical
structure accounted for 48% in fresh cells, and 59.5%
in frozen cells.
The survival rate of Ⅰt
group was 16/17, which was significantly higher than that of Ⅰc
group (3/9) (P=0.0022,
exact
probablility). The AHF rats showed low consciousness and appetite, but the situation
in the transplant group was a little better than that in the control group.
Twelve rats of Ⅱt
group and 16 of Ⅱc
group died before sacrifice. Varying degrees of light yellow ascites were found
by autopsy in the dead rats. The remaining livers of 10 out of 13 sacrificed rats
in the Ⅱt
group and of 6 out of 8
sacrificed rats in the Ⅱc
group were weighed 2 days
after the 80% hepatectomy. The
same procedures were performed in another set of rats and in 10 normal rats 7
days after the 80% hepatectomy. The weights of remaining livers in the Ⅱt
and Ⅱc
groups were 6.34±0.88g
(n=10) and 6.22±0.95g
(n=6 respectively 2 days after the 80% hepatectomy, and 8.80±1.02g (n=10) and 8.22±0.50g (n=3) respectively
7 days after the 80% hepatectomy. There was no significant difference
between the Ⅱt
and Ⅱc
groups (P＞0.05),
but
the weights of livers at 7 days in both groups were significantly lower than
that of the normal rats (13.27±1.99g, n=10) (P＜0.01). Histological
examination revealed that the structure of the remaining livers was
normal. The results of the hepatic function were showed in the Table 1.
Table 1
Assay of biochemical values of sera after liver failure
(x-±s)
| Ⅱt group | Ⅱc group | Normal rats | |
| 2 days after liver faliure | |||
| ALB (g/L) | 23.62±3.69 (13) | 24.13±3.56 (8) | 33.87±2.36a (15) |
| TBr (μmol/L) | 20.30±13.23 (13) | 24.00±17.22 (8) | 2.29±1.61a (15) |
| DBr (μmol/L) | 12.89±9.12 (13) | 15.61±13.63 (8) | 0.40±0.39a (15) |
| 7 days after liver failure | |||
| ALB (g/L) | 30.00±4.22 (12) | 26.00±3.00 (3) | 33.87±2.36a (15) |
| TBr (μmol/L) | 2.97±1.02 (11) | 2.90±0.36 (3) | 2.29±1.61 (15) |
| DBr (μmol/L) | 1.14±0.49 (11) | 0.80±0.56 (3) | 0.40±0.39 (15) |
Note:
Ⅱt
group: transplant group in the part two; Ⅱc
group: control group in the part two; Numbers in parentheses are numbers of
rats.aP＜0.01,
Ⅱt
group vs
Ⅱc
group, and Ⅱc group vs
mormal rats.
DISCUSSION
The survival rate (16/17) of the AHF rats after the transplantation was significantly
higher than that of the Ⅰc
group (3/9), and the living situation
of the former during the survival was apparently superior to that of the latter.
We found that after the 30th day of cryopreservation in liquid nitrogen, the
viability of hepatocytes no longer decreased in subsequent freeze up to 180
days, and the appearance and function of the hepatocytes
remained well［3］.
In view of the above, it is possible to cure AHF using pighepatocytes
cryopreserved for 150 days.
The transplanted cells will be
destroyed by immunological rejection. To enable the grafted porcine hepatocytes
to play the therapeutic role in the body of a xenogeneic animal, we adopted the
transplanting method of before and after the 80% hepatectomy respectively.
Moreover, as hypothermia is able to reduce the immunogenicity
of cells［4］,
and thus delaying or abating the rejection, the hepatocytes
can gain more time to recover their functions. The conformational investigation
of membrane proteins revealed that the content of αhelical structure increased from 48% to 59.5%
after freeze. This result was possibly
responsible for the report that hypothermia was able to reduce the
immunogenicity of cells. The conformational change of membrane proteins might
subsequently
affect the characteristics of their functioning domains.
The possible mechanism of
treatment of AHF with hepatocyte transplantation was thought to be that the
transplanted hepatocytes produce hepatocyte growth factor which promotes liver
regeneration and recovery of liver function, and the cells act as a substitution
for the AHF liver. From our experiment, ALB concentrations of
the Ⅱt
and Ⅱc
groups 2 days after the 80%
hepatectomy
were significantly lower than that of the normal rats. TBr and DBr increased
rapidly, but there was no significant difference between
the Ⅱt and
Ⅱc groups,
neither was there any significant
difference in the weight of remaining livers between the two
groups 2 and 7 days after the 80% hepatectomy. However, the survival rate of the
transplant group was significantly higher than that of the control group. The
results suggest that the mechanism of treating AHF with hepatocyte
transplantation may not only be the effects of the substitution for the liver
and the hepatocyte growth factor, but also be the effect of other unknown
factors. It may be
a result of multiple effects.
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1Department
of Surgery, Third Hospital, Beijing Medical University,
Beijing 100083, China.
2Department of
Biological Sciences and Biotechnology, Tsinghua University, Beijing
100084, China.
*Supported by the Science
Foundation of Chinese Ministry of Health
(No. 942194) and the National
Natural Science Foundation of China (No.
59493208)
Corespondence to Dr. Lin Cong,Department
of Surgery, Third Hospital, Beijing Medical University,
Beijing 100083, China.
Tel.+86·10·62017691
Ext. 2547.
Received
30th
July 1996.