永生化HepGL肝细胞移植救治大鼠急性肝衰竭模型的实验研究

发布时间：2018-08-02 17:56

【摘要】：研究背景：我国是肝炎大国,每年死于肝衰竭的患者约有40万人。肝衰竭的内科治疗效果较差,在原位肝移植(orthotopic liver transplantation,OLT)临床应用以前,肝衰竭的的存活率20%。目前,国外文献报道经有效治疗的肝衰竭患者的存活率65%。但国内肝衰竭患者的预后仍很不理想。OLT治疗肝衰竭的效果理想而肯定,是目前治疗急性肝衰竭、终末期肝病和代谢性肝病的主要方法。但由于供体短缺、免疫排斥及高额的手术治疗费用严重限制其应用于临床,许多患者在等待中死亡。目前治疗肝衰竭的新兴方法还有人工肝支持系统、肝细胞移植、异种器官移植等。其中肝细胞移植由于其众多的优点,已被认为是最具前景的AHF治疗方法之一。肝细胞移植相较于原位肝移植具有以下优点：①相对OLT巨大的手术创伤而言,对患者的创伤性损害较小,尤其是已经不能耐受手术的肝功能极差的患者,同时也可以避免OLT所引起的急性血管排斥反应；②操作方法简单,目前主要采用门静脉注入方式,其他还有脾内注射,大网膜种植等移植方法；③手术费用低,是全肝移植费用的5%-10%；④分离的肝细胞可以保存,所以可以是随时的、多次重复的用于肝衰竭病人的治疗；⑤分离一次可以用于多个肝衰竭患者的治疗,即一对多的治疗；⑥肝细胞移植可保证患者肝脏结构的完整性,急性肝衰竭患者的肝脏可渡过危险期,通过再生使其自身功能的恢复；⑦可对移植的肝细胞进行基因修饰。目前正在研究可供移植的肝细胞有多种,如异种肝细胞、人体干细胞和原代肝细胞移植。异种移植利用猪和狒狒等灵长类动物肝脏作为细胞来源,具有来源丰富,繁育方便,价格较低等优点。但异种移植带来的免疫排斥反应、生理生化兼容性及潜在的病原体传播的风险阻碍了其在临床中的应用。限于动物种系选择、病原体传播及伦理等方面,异种肝细胞移植较其他两种移植有较大的风险。干细胞具有自我更新和分化能力,分有胚胎肝细胞、胎儿干细胞和成人干细胞。源于胚胎和胎儿的干细胞受到政治法律和伦理道德的限制,即使具有功能良好和免疫排斥少等优势也未能大规模展开应用。研究证明人体肝脏祖细胞能修复受损肝脏并使肝脏再增殖,但移植后细胞难以跟踪证明是否能使肝脏完全再生。另有骨髓干细胞和脂肪间充质干细胞具有细胞融合和多向分化能力,成为细胞移植研究热点。肝细胞移植是由Bumgardner率先提出,而世界第一例人体肝细胞移植临床应用是Mito于1992年分离慢性肝病患者自身肝细胞并进行自体移植,证明人肝细胞有修复急性肝损伤的能力,可在一定条件下重建衰竭肝脏。同时有文献报道表明有急性肝衰竭患者接受人肝细胞移植,移植后血氨和胆红素水平降低,并且部分患者肝功能完全恢复。肝细胞移植作为新的治疗AHF手段前景诱人,但还需要克服的问题仍很多。如肝细胞移植后引发的免疫排斥反应就是一个重要问题,将移植肝细胞进行微囊包裹,或者从肝脏实质细胞中去除抗原递呈细胞可以降低肝细胞免疫源性,或者封闭抗原递呈细胞的T细胞共刺激分子(B7蛋白)也可以调控免疫源性,这些都是解决免疫问题的措施。如何解决免疫排斥反应是肝细胞移植应用的重点,但如何提供足够数量、功能良好肝细胞更是决定肝细胞移植能否广泛应用的关键。相比全肝移植,肝细胞易于保存和运输,若能解决肝细胞的来源问题,肝细胞移植将来可在临床上得到的广泛应用。研究目的：本实验通过向SD大鼠脾内注射移植永生化HepGL肝细胞,对急性肝衰竭的大鼠模型进行救治,研究永生化HepGL肝细胞在动物体内的功能,探讨其是否具有替代并重建衰竭肝脏的能力。同时为其将来作为生物人工肝的种子细胞的应用奠定理论基础。实验方法： 1.SD大鼠急性肝衰竭(Acute Hepatic Failure, AHF)模型的建立；采用90%肝部分切除术建立SD大鼠AHF模型,具体步骤如下： (1)术前准备包括,术前SD大鼠禁食6h,改喂饮10%葡萄糖水；手术器械高压灭菌；药物(硫酸阿托品、乙醚、青霉素钠等),耗材(5-0丝线、纱布、棉球、棉签、一次性使用注射器等)等的准备； (2)术前准备完成后,将大鼠用乙醚麻醉,待大鼠麻醉后,将其固定于自制手术台上,肌注阿托品0.03mmg(配成0.5ml盐水溶液),并用50ml离心管内放入浸湿了的无水乙醚的棉球,将管口对准大鼠口鼻,持续吸入麻醉,麻醉深度的调节以大鼠停止躁动,呼吸平稳为准； (3)一切就绪后,备皮刀刮毛、腹部常规3次消毒、铺无菌洞巾； (4)选择腹部横切口入腹,以剪刀剪开皮肤、肌肉及腹膜各层,以自制拉钩充分暴露肝脏,解剖各肝叶间及与周围组织之间的菲薄韧带； (5)依次游离出各肝叶后,在右上叶(Right superior lobe,RSL)与肝中叶(Median lobe,ML)之间,游离出肝脏的Glisson系统头支主干,以5-0丝线穿过结扎,可见肝中叶(ML)、左外叶(Left lateral lobe,LLL)迅速缺血,待肝叶变为土黄色后,5-0丝线结扎相应肝叶的肝蒂后并切除；游离出右上叶(RSL)与右下叶(Right inferior lobe,RIL)的共同Glisson系统右支主干,5-0丝线结扎后同上依次切除右上叶与右下叶；仅保留包括前叶(Superior caudate lobe,SCL)与后叶(Inferior caudate lobe,ICL)的尾状叶(Caudate lobe,CL)及腔静脉旁部(paracaval liver,PL)(约占肝脏总量的10%)。检查腹腔内无出血后,于腹腔内注射青霉素20万单位溶液lml,依次缝合腹膜、肌层和皮肤,缝合皮肤前停止乙醚麻醉； (6)术后普通块料喂养,喂饮10%葡萄糖水,室温18～22℃,光照12h/d。 2.永生化HepGL肝细胞移植； (1)实验分组：实验组(n=21)—脾内注射约2.5*107个永生化HepGL肝细胞(配成0.5mlDMEM混悬液)/只；空白组(n=21)—未做任何治疗的急性肝衰竭组； (2)实验组永生化HepGL肝细胞移植步骤： SD大鼠腹部行约1cm纵切口,用无菌棉签伸入胃下提出脾脏,2.5*107个永生化HepGL肝细胞(配成0.5mlDMEM混悬液)大鼠脾内注射,注射24h后SD大鼠行90%肝部分切除术。 3.观察项目； (1)大体观察：观察空白组及实验组SD大鼠术后的饮食、精神状态、活动量及对外界刺激反应及相关的症状表现等情况。 (2)存活率：记录大鼠的死亡时间。 (3)生化检查：收集Oh、24h、72h、7d、14d大鼠的静脉血,检测肝功能指标包括：①谷丙转氨酶(ALT)、②总胆红素(Tbil)、③白蛋白(ALB)、④血氨(NH3)、⑤血糖(Glu)、⑥凝血时间(PT)等的变化。 (4)病理组织检查：在24h、72h、7d、14d取大鼠肝脏及脾脏做组织病理切片及免疫组化,以了解大鼠肝脏急性肝衰竭病理变化、恢复情况以及移植的HepGL细胞在大鼠体内的存活的情况。实验结果： 1.大体观察实验组与空白组SD大鼠均在术后3-10min内即可苏醒翻身爬起,空白组大鼠12h后出现急性肝衰竭症状,包括精神萎靡,拒饮食,对外界反应迟钝,全身蜷缩,鼠毛竖立,尿色发黄、腹泻,口鼻眼角出血等,大部分个体开始逐渐出现肝昏迷直至死亡。实验组大鼠12h后也有精神萎靡,食欲下降,对外界反应迟钝,全身蜷缩,鼠毛竖立等症状,但程度较轻,且无口鼻眼角出血等症状,也有部分个体出现肝昏迷并死亡,部分个体尸解可见腹腔内有较清澈的腹水。 2.存活率观察两组术后7d存活率,实验组7d存活率66.67%(14/21),死亡时间点分别为15h、35h、41h、45h、60h、62h;空白组7d存活率为28.57%(6/21),死亡时间点分别为16h、21h、23h、26h、32h、35h、38h、38h、41h、42h、50h、54h、60h、63h、70h,两组死亡基本全部发生在3d内,大鼠存活超过3d,便可长期存活。 3.生化检查两组大鼠术后各生化指标均有明显变化,两组ALT、Tbil、NH3、PT等四项指标均在术后24h上升到最高值,且四项指标两组之间均存在差异(P0.05),后逐渐恢复；两组ALT、Tbil指标72h也存在明显差异(P0.05),在7d、14d点基本持平；两组PT仅在24h有明显差异；两组ALB、Glu术后均出现下降,ALB指标空白组在7d出现最低值,而实验组在72h出现最低值,两组在24h、72h、7d各点均有明显差异(P0.05); Glu指标两组均在24h出现最低值,在72h有明显差异(P0.05)。 4.病理组织检查空白组大鼠术后24h时见残余肝脏大体组织色泽淡黄,包膜肿胀,质地松软,触之易碎,镜下可见空泡变性和炎细胞浸润；72h时残余肝脏较前增大,边缘可见血管增生,镜下可见弥漫大量空泡变性和炎细胞浸润,肝小叶结构破坏。实验组大鼠注射后24h时,脾脏红肿,呈鲜红色,术后肝脏病理表现同空白组,脾脏可见表面有斑点状突起。免疫组化可见细胞在脾脏内成团聚集,在肝脏内主要停留在肝血管窦部。实验结论：实验组大鼠生存质量以及存活率明显优于空白组,两组生化指标在一定时间点存在明显差异,组织病理发现实验组大鼠体内存在移植的永生化HepGL肝细胞；说明永生化HepGL肝细胞在动物体内具备一定的生物学功能,能一定程度的替代部分肝功能,可作为生物人工肝的种子细胞研究使用。
[Abstract]:Research background:
China is a great country of hepatitis, about 400 thousand people die of liver failure every year. The medical treatment effect of liver failure is poor. The survival rate of liver failure was 20%. before the clinical application of orthotopic liver transplantation (OLT). The survival rate of patients with liver failure treated by effective treatment was 65%. but domestic liver failure was reported in foreign literature. The prognosis of the patients is still not ideal for the treatment of liver failure with ideal.OLT. It is the main method for the treatment of acute liver failure, end-stage liver disease and metabolic liver disease. However, due to the shortage of donor, immune rejection and high cost of surgical treatment, it severely restricts its application and many patients are waiting to die. Currently, the treatment of liver failure is in the treatment of liver failure. The new methods of exhaustion are artificial liver support system, hepatocyte transplantation, xenotransplantation and so on. Hepatocyte transplantation has been considered as one of the most promising methods of AHF treatment because of its many advantages. The injury damage is small, especially the patients who have been unable to tolerate the poor liver function of the operation, and can also avoid the acute vascular rejection caused by OLT; 2. The operation method is simple, the main use of portal vein injection, the other methods such as intrarenic injection and greater omentum implantation, and the low cost of operation are all liver transplantation. The 5%-10% of the cost; (4) the isolated liver cells can be preserved so that they can be kept at any time and repeated for the treatment of patients with liver failure; (5) separation one time can be used to treat patients with multiple liver failure, that is, one to many treatments; 6. Hepatocyte transplantation can guarantee the integrity of the patients' liver structure and the liver of patients with acute liver failure. After the critical period, the function of the transplanted hepatocytes can be recovered by regeneration.
There are many kinds of liver cells that can be transplanted, such as xenoliver cells, human stem cells and primary hepatocyte transplantation. Xenotransplantation of primate liver, such as pigs and baboons, as cell source, has the advantages of rich source, convenient breeding and low price. However, the immune rejection, physiological and biochemical compatibility of different species of transplantation The risk of sexual and potential pathogen transmission hinders its clinical application. Xenotransplantation is more risky than the other two types of transplantation, limited to animal species selection, pathogen transmission and ethics. Stem cells have the ability to renew and differentiate themselves, including embryonic liver cells, fetal stem cells and adult stem cells. The stem cells of embryos and foetuses are limited by political, legal and ethical standards. Even if they have the advantages of good function and less immune rejection, the human liver progenitor cells can repair damaged liver and regenerate the liver, but the cells after transplantation can not be tracked to prove that the liver can be completely regenerated. Bone marrow stem cells and adipose mesenchymal stem cells have the ability of cell fusion and multidifferentiation, which has become a hot spot in cell transplantation research. Hepatocyte transplantation is first proposed by Bumgardner. The first case of human hepatocyte transplantation in the world is the isolation of liver cells from chronic liver diseases in 1992 by Mito and autologous transplantation. Liver cells have the ability to repair acute liver damage and can reconstruct the liver under certain conditions. There are reports that patients with acute liver failure receive human hepatocyte transplantation, blood ammonia and bilirubin levels decrease after transplantation, and some patients have complete recovery of liver function.
Hepatocyte transplantation is attractive as a new method for the treatment of AHF, but there are still a lot of problems to be overcome. For example, the immune rejection after hepatocyte transplantation is an important problem. It is an important problem to encapsulate the transplanted liver cells in microcapsules or remove the antigen presenting cells from the liver parenchyma to reduce the immunogenicity of liver cells, or to seal the liver cells. The T cell co stimulator (B7 protein) of the antigen presenting cell can also regulate the immunity. These are all measures to solve the immune problems. How to solve the immune rejection is the key point of the application of liver transplantation, but how to provide sufficient quantity and good function of liver cells is the key to determine whether the liver cell transplantation can be widely used. Hepatocytes can be easily preserved and transported in whole liver transplantation. If the source of hepatocytes can be solved, hepatocyte transplantation will be widely used in clinic in the future.
The purpose of the study is:
In this experiment, immortalized HepGL hepatocytes were injected into the spleen of SD rats, and the rat model of acute liver failure was treated, and the function of immortalized HepGL hepatocytes in the animals was studied, and the ability to replace and reconstruct the liver failure was explored. On the basis.
Experimental methods:
1. Establishment of acute hepatic failure (AHF) model in SD rats, and establishment of AHF model in SD rats by 90% partial hepatectomy, the specific steps are as follows:
(1) pre operation preparation included preparation of pre operation SD rats fasting 6h, feeding 10% glucose water, high pressure sterilization of surgical instruments, drugs (atropine sulfate, ether, penicillin sodium, etc.), materials (5-0 silk thread, gauze, cotton ball, cotton swab, disposable syringe etc.).
(2) after the preparation was completed before the operation, the rats were anesthetized with ether, and after the rats were anaesthetized, the rats were fixed on the self-made operating table. The atropine 0.03mmg (with 0.5ml saline solution) was injected into the rat, and the soaked anhydrous ether cotton ball was put into the 50ml centrifuge tube. The mouth was aimed at the rat's mouth and nose, and the anesthetic depth was adjusted to stop the rats. Move, the breath is steady;
(3) when all is ready, skin preparation is done, and abdominal routine is sterilized for 3 times.
(4) a transverse incision in the abdomen was chosen to cut off the skin, muscles and each layer of the peritoneum with a scissors. The liver was fully exposed with a self-made retractor, and the thin ligaments between the leaves of the liver and the surrounding tissues were dissected.
(5) after dissociating the liver leaves in turn, between the right upper lobe (Right superior lobe, RSL) and the middle lobe of the liver (Median lobe, ML), the head of the head of the liver Glisson system was dissociated, the middle lobe of the liver (ML), the left outer leaf (Left lateral lobe,) were rapidly ischemic, and the liver leaf became yellow, and the 5-0 silk thread ligated the liver of the liver. The right branch of the right branch of the right upper right lobe (RSL) and the right lower lobe (Right inferior lobe, RIL) was removed from the right branch of the right branch of the right upper lobe (Right inferior lobe, RIL). The upper right upper lobe and right lower lobe were excised in turn after the ligation of the 5-0 silk thread. Paracaval liver (PL) (about 10% of the total amount of liver). After examination of no hemorrhage in the abdominal cavity, the 200 thousand unit solution of penicillin was injected into the abdominal cavity. The peritoneum, the muscle layer and the skin were sutured in turn, and the ether anesthesia was stopped before suturing the skin.
(6) postoperative common lump feeding, feeding 10% glucose water, room temperature 18~22 degrees, light 12h/d.
2. immortalized HepGL hepatocyte transplantation;
(1) experimental grouping:
In the experimental group (n=21), about 2.5*107 immortalized HepGL hepatocytes (0.5mlDMEM suspension) were injected into the spleen.
Blank group (n=21) - acute liver failure group without any treatment;
(2) the procedure of immortalized HepGL hepatocyte transplantation in the experimental group:
SD rats were treated with a 1cm longitudinal incision, and the spleen was put under the stomach with aseptic cotton swabs, and 2.5*107 immortalized HepGL hepatocytes (with 0.5mlDMEM suspension) were injected into the spleen of the rat, and the 90% partial hepatectomy was performed in the SD rats after the injection of 24h.
3. observation project;
(1) General observation: To observe the diet, mental state, activity, external stimulus response and related symptoms of SD rats in blank group and experimental group after operation.
(2) survival rate: record the time of death in rats.
(3) biochemical examination: the venous blood of Oh, 24h, 72h, 7d, 14d rats was collected, and the indexes of liver function included: (1) the changes of glutamic pyruvine aminotransferase (ALT), total bilirubin (Tbil), albumin (ALB), blood ammonia (NH3), blood glucose (Glu), and blood coagulation time (PT).
(4) pathological examination: histopathological section and immunohistochemistry of rat liver and spleen were taken in 24h, 72h, 7d, and 14d to understand the pathological changes of liver acute liver failure in rats, the recovery and the survival of the transplanted HepGL cells in rats.
Experimental results:
1. general observation
The experimental group and the blank group SD rats were all able to wake up and climb up in 3-10min after the operation. The rats in the blank group had the symptoms of acute liver failure after 12h, including the mental retardation, the refusal of diet, the outside reaction, the whole body curling, the erect hair of the rat hair, the yellow color, the diarrhea, the nose and the corner of the eye, and so on, and most of the individuals began to gradually appear liver coma until death. After 12h, the rats in the experimental group were also mentally retarded, the appetite declined, the external reaction was slow, the whole body curled up, the rat hair erected, but the degree was mild, and there was no nasal angle bleeding and other symptoms. Some individuals appeared liver coma and died. Some individuals showed that there were clearer ascites in the abdominal cavity.
2. survival rate
The survival rate of 7D in the two groups was 66.67% (14/21), and the time of death was 15h, 35h, 41h, 45h, 60H, 62H, and the survival rate of the 7d was 28.57% (6/21). Long term survival.
3. biochemical examination
The biochemical indexes of the two groups were obviously changed. The four indexes of the two groups, such as ALT, Tbil, NH3, PT, were all increased to the highest value after the operation, and the four indexes were different between the two groups (P0.05), and then gradually recovered; the two group ALT, Tbil index 72h also had obvious differences (P0.05), in 7d, the 14d points were basically flat; the two groups were only significantly worse than the two groups. The two groups of ALB, Glu were all decreased after the operation, the ALB index blank group had the lowest value in 7d, while the experimental group had the lowest value in 72h. The two groups were significantly different at 24h, 72h, 7d (P0.05), and the Glu index two groups were all lowest in 24h, and there were obvious differences in 72h.
4. pathological tissue examination
In the blank group, the residual liver tissues were pale yellow, swollen, soft, soft, fragile, vacuolated degeneration and infiltration of inflammatory cells under the microscope, while the residual liver was enlarged at 72h and vascular proliferation was visible at the edge of the liver. The diffuse large number of vacuolar degeneration and infiltration of inflammatory cells and the destruction of hepatic lobule structure were seen under the microscope. The rat liver lobule structure was destroyed. Experimental group rats were found under the microscope. At 24h after injection, the spleen was red and swollen and bright red. The pathological manifestations of the liver after the operation were in the same blank group, and the spleen was spotted on the surface. The immuno histochemistry showed that the cells were clustered in the spleen and stayed in the hepatic vascular sinus mainly in the liver.
Experimental conclusions:
The survival quality and survival rate of the experimental group were obviously better than that of the blank group. There were obvious differences between the two groups of biochemical indexes at a certain time point. The histopathology found the transplanted immortalized HepGL hepatocytes in the experimental group. It showed that the immortalized HepGL hepatocyte had certain biological functions in the animal body and could be replaced to a certain extent. Some liver functions can be used as seed cells for bioartificial liver.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R657.3

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