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Research on liver regeneration driven by the amniotic membrane

Xu, Jia; Zhang, Haitao; Li, Ji; Li, Ning

doi: 10.3760/cma.j.issn.0366-6999.20131632
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Beijing You An Hospital, Capital Medical University, Beijing 100069, China (Xu J, Zhang HT, Li J and Li N)

Department of General Surgery, Beijing University of Chinese Medicine Third Affiliated Hospital, Beijing 100029, China (Xu J) Correspondence to: Prof. Li Ning, Beijing You An Hospital, Capital Medical University, Beijing 100069, China (Email: liningbjyah@vip.sina.com)

This study was supported by the Beijing Municipal Science and Technology Commission (Nos. D09050703560902 and D09050703560903).

(Received June 23, 2013)

Edited by Hao Xiuyuan

Primary liver cancer is one of the most common cancers in China. Among all liver cancers, more than 90% of the cases are hepatocellular carcinoma (HCC) with a mortality of 20.40/0.1 million people. In general, surgery is the first-line treatment for primary liver cancer. With the development of diagnostic procedures, surgical technologies, and perioperative treatments, the mortality and complications of HCC have decreased. However, compared to other surgeries, the postoperative complications of hepatectomy have remained at relatively high levels. Of these complications, liver failure is one of the most important and common complications, and may lead to death. Functional protection after hepatectomy is crucial for the postoperative treatment and rehabilitation of patients. Derived from the cytotrophoblast, the amniotic membrane (AM) is the inner membrane of the fetal membranes. Recent research has confirmed its function to secrete basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and to inhibit the mRNA expression of transforming growth factor (TGF)-β. It also causes no expression of amniotic HLA-II and low expression of HLA-I. Owing to the mild rejection reactions, sufficient supply, and lack of associated ethical problems, AM implantation has become a hotspot in liver regeneration research. In this study, we believe that AM implantation could shorten the liver recovery period, and minimize the possibility of liver failure, which makes the larger-volume liver surgery possible.

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METHODS

AM preparation

AMs were obtained from the placenta of healthy pregnant woman with abdominal delivery. Negativity for serumtransmitted diseases, such as HIV, HBV, HCV, and syphilis, was confirmed by prior testing. The blood was removed from the membrane by soaking it in phosphate-buffered saline (PBS) solution for 15 minutes. Subsequently, the AMs were soaked in antibacterial physiological saline containing 50 mg/L penicillin, 50 mg/L streptomycin, 100 mg/L neomycin, and 2.5 mg/L amphotericin B. The AMs were peeled off in a cleaned operation desk and shaped into pieces of 3 cm×3 cm.

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Sample distribution and treatment

One hundred and twenty male rats were prepared according to the liver regeneration model of 70% liver ablation. The samples were divided into three groups of 40 rats each: (1) Control group, with 70% liver hepatectomy; (2) AM-implanted group, a 3 cm×3 cm AM was implanted in the muscle layer of the abdominal wall after partial hepatectomy (PH); and (3) PHGF-injected group, injection of 2 mg/d hepatocyte growth-promoting factor (PHGF) sodium solution (2 ml/d) in the tail vein after PH. At 12, 24, 48, and 72 hours post-surgery, blood samples of all the rats were examined by enzyme-linked immunosorbent assay (ELISA) for their HGF, EGF, and TGF-β levels. Fresh liver tissue was examined for the cell cycle by flow cytometric analysis. Other tissue samples were observed by hematoxylin-eosin (HE) staining after 10% formalin fixation for the overall situation. The expression of proliferating cell nuclear antigen (PCNA) was examined by immunohistochemistry.

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Statistical analysis

Data are presented as the mean±standard deviation (SD). All data were analyzed by the rank sum test using SPSS15.0 software (SPSS Inc., IL, USA). Values of P <0.05 were considered to indicate significant differences.

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RESULTS

PCNA values

By 12–24 hours after PH, the PCNA values in the PHGF-injected group and AM-implanted group were higher than that in the control group (Table 1). The PCNA values in the PHGF-injected group were significantly higher than that in the AM-implanted group and control group (P=0.032). At 24–48 hours after PH, the PCNA index in the AM-implanted group started to increase and became significantly higher than those in the PHGF-injected group and control group (P=0.028; Figure 1A-C).

Table 1

Table 1

Figure 1.

Figure 1.

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Results of flow cytometric analyses

At 48 hours after PH, the PI values in the PHGF-injected group and AM-implanted group were much higher than those in the control group, with a significant difference (P=0.034. Table 1). Moreover, the PI in the PHGF-injected group was higher than that in the AM-implanted group. At 72 hours, the PI in the AM-implanted group was higher than that in the PHGF-injected group and control group, with a significant difference (P=0.017), while no significant difference was observed between the PHGF-injected group and control group (P=0.058; Figure 1D-1F).

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Serum HGF, EGF, and TGF-β levels

The HGF levels in the PHGF-injected group and AM-implanted group started to increase rapidly after PH, and remained higher than that in the control group with a significant difference (P=0.021). At 72 hours, the level in the AM-implanted group was higher than that in the PHGF-injected group. The EGF levels in all samples started to decrease after PH, with no correlations with the PI and PCNA-positive index among the three groups. The TGF-β levels in the PHGF-injected group and control group started to decrease and then increased after PH, while the TGF-β levels in the AM-implanted group remained at low levels, with a significant difference (P <0.043; Figure 1G-1I).

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DISCUSSION

As the inner membrane of the fetus membranes, the AM is derived from the cytotrophoblast. Current researchers believe that its functions are secretion of bFGF, EGF, HGF, and KGF and inhibition of TGF-β mRNA expression. Meanwhile, the AM does not express HLA-II antibodies or HLA-I, and suffers little rejection. Moreover, it has plenitudinous supply, and lack of associated ethical problems, the sufficient supply promotes the application of AM in clinical practice.1,2

According to previous studies, the DNA formation in liver cells of hepatectomy rats can be divided into a pre-reproduction stage (12–14 hours after hepatectomy) and a production stage (14–36 hours after hepatectomy). Although there are no clear criteria for these two stages, within 12 hours of the surgery, the majority of the liver cells existed in G0 stage, and then transformed into G1 stage, followed by cell karyokinesis and reproduction The reproduction reached the highest point by 24 hours after surgery. In normal rats, the liver weight increased quickly, especially by 3–4 days after the surgery, and recreated the normal weight by 7 days after the surgery.

In this study, the PCNA-positive index and PI in the PHGF-injected group and AM-implanted group were clearly increased by 12–48 hours after PH, while the PCNA-positive index and PI in the AM-implanted group were lower than those in the PHGF-injected group. However, by 72 hours, the indexes in the AM-implanted group were higher than in the PHGF-injected group. At that time, the PCNA-positive index and PI index in the PHGF-injected group and control group showed no significant differences. The HGF, EGF, and TGF-β levels in the samples presented the following features: (1) the HGF levels in the PHGF-injected group and AM-implanted group increased quickly after PH, with significant differences from the control group by 72 hours after PH, while the increases in the AM-implanted group were higher than those in the PHGF-injected group; (2) there were no obvious correlations of the EGF level and the PCNA-positive index and PI in any of the groups; and (3) for all the samples, the TGF-β levels within 72 hours showed a tendency to decrease and then increase, while the TGF-β levels in the AM-implanted group remained at low levels and were lower than those in the PHGF-injected group and control group.

Based on our analyses of the data, we speculate that the AM was able to activate the liver regeneration through HGF secretion, which produced a better effect than PHGF injection. The HGF, EGF, and TGF-β levels observed in the study and the liver regeneration mechanism helped us to conclude that the liver regeneration depended not only on the HGF secretion, but also on the TGF-β control and DNA reproduction, which could be a new theory in liver regeneration.

Although this study has led to new understandings, we still identified more details that require further discussion. For example, the aspect of whether functional cells like those producing HGF and TGF-β or controlled by the AM could be adjusted by the body's modulatory factors. We believe that further trials should be performed to confirm these issues.

In this study, the PCNA-positive index and PI in the PHGF-injected group and AM-implanted group were clearly increased by 12–48 hours after PH, while the PCNA-positive index and PI in the AM-implanted group were lower than that in the PHGF-injected group. However, by 72 hours, the indexes in the AM-implanted group were higher than in the PHGF-injected group. At that time, the PCNA-positive index and PI index in the PHGF-injected group and control group showed no significant differences. The HGF levels in the AM-implanted group and PHGF-injected group were much higher than in the control group. No obvious correlations were observed between EGF, PI, and PCNA-positive index among the three groups. The consistently low TGF-β levels in the AM-implanted group differed significantly from those in the PHGF-injected group and control group. The TGF-β levels in the PHGF-injected group and control group showed no obvious difference and gradually increased.

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REFERENCES

1. Zamegar R, Michalopoulos GK. The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis. J Cell Biol 1995; 129: 1177-1180.
2. Deolinda de Oliveira Pena J, Melo GB, Gomes JA, Haapalainen EF, Komagome CM, Santos NC, et al. Ultrastructural and growth factor analysis of amniotic membrane preserved by different methods for ocular surgery (in Portuguese). Arq Bras Oftalmol 2007; 70: 756-762.
Keywords:

amniotic membrane; liver regeneration; partial hepatectomy; proliferation index

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