【摘要】：研究背景： 我國是肝炎大國,每年死于肝衰竭的患者約有40萬人。肝衰竭的內(nèi)科治療效果較差,在原位肝移植(orthotopic liver transplantation,OLT)臨床應(yīng)用以前,肝衰竭的的存活率20%。目前,國外文獻(xiàn)報(bào)道經(jīng)有效治療的肝衰竭患者的存活率65%。但國內(nèi)肝衰竭患者的預(yù)后仍很不理想。OLT治療肝衰竭的效果理想而肯定,是目前治療急性肝衰竭、終末期肝病和代謝性肝病的主要方法。但由于供體短缺、免疫排斥及高額的手術(shù)治療費(fèi)用嚴(yán)重限制其應(yīng)用于臨床,許多患者在等待中死亡。目前治療肝衰竭的新興方法還有人工肝支持系統(tǒng)、肝細(xì)胞移植、異種器官移植等。其中肝細(xì)胞移植由于其眾多的優(yōu)點(diǎn),已被認(rèn)為是最具前景的AHF治療方法之一。肝細(xì)胞移植相較于原位肝移植具有以下優(yōu)點(diǎn)：①相對OLT巨大的手術(shù)創(chuàng)傷而言,對患者的創(chuàng)傷性損害較小,尤其是已經(jīng)不能耐受手術(shù)的肝功能極差的患者,同時(shí)也可以避免OLT所引起的急性血管排斥反應(yīng)；②操作方法簡單,目前主要采用門靜脈注入方式,其他還有脾內(nèi)注射,大網(wǎng)膜種植等移植方法；③手術(shù)費(fèi)用低,是全肝移植費(fèi)用的5%-10%；④分離的肝細(xì)胞可以保存,所以可以是隨時(shí)的、多次重復(fù)的用于肝衰竭病人的治療；⑤分離一次可以用于多個(gè)肝衰竭患者的治療,即一對多的治療；⑥肝細(xì)胞移植可保證患者肝臟結(jié)構(gòu)的完整性,急性肝衰竭患者的肝臟可渡過危險(xiǎn)期,通過再生使其自身功能的恢復(fù)；⑦可對移植的肝細(xì)胞進(jìn)行基因修飾。 目前正在研究可供移植的肝細(xì)胞有多種,如異種肝細(xì)胞、人體干細(xì)胞和原代肝細(xì)胞移植。異種移植利用豬和狒狒等靈長類動(dòng)物肝臟作為細(xì)胞來源,具有來源豐富,繁育方便,價(jià)格較低等優(yōu)點(diǎn)。但異種移植帶來的免疫排斥反應(yīng)、生理生化兼容性及潛在的病原體傳播的風(fēng)險(xiǎn)阻礙了其在臨床中的應(yīng)用。限于動(dòng)物種系選擇、病原體傳播及倫理等方面,異種肝細(xì)胞移植較其他兩種移植有較大的風(fēng)險(xiǎn)。干細(xì)胞具有自我更新和分化能力,分有胚胎肝細(xì)胞、胎兒干細(xì)胞和成人干細(xì)胞。源于胚胎和胎兒的干細(xì)胞受到政治法律和倫理道德的限制,即使具有功能良好和免疫排斥少等優(yōu)勢也未能大規(guī)模展開應(yīng)用。研究證明人體肝臟祖細(xì)胞能修復(fù)受損肝臟并使肝臟再增殖,但移植后細(xì)胞難以跟蹤證明是否能使肝臟完全再生。另有骨髓干細(xì)胞和脂肪間充質(zhì)干細(xì)胞具有細(xì)胞融合和多向分化能力,成為細(xì)胞移植研究熱點(diǎn)。肝細(xì)胞移植是由Bumgardner率先提出,而世界第一例人體肝細(xì)胞移植臨床應(yīng)用是Mito于1992年分離慢性肝病患者自身肝細(xì)胞并進(jìn)行自體移植,證明人肝細(xì)胞有修復(fù)急性肝損傷的能力,可在一定條件下重建衰竭肝臟。同時(shí)有文獻(xiàn)報(bào)道表明有急性肝衰竭患者接受人肝細(xì)胞移植,移植后血氨和膽紅素水平降低,并且部分患者肝功能完全恢復(fù)。 肝細(xì)胞移植作為新的治療AHF手段前景誘人,但還需要克服的問題仍很多。如肝細(xì)胞移植后引發(fā)的免疫排斥反應(yīng)就是一個(gè)重要問題,將移植肝細(xì)胞進(jìn)行微囊包裹,或者從肝臟實(shí)質(zhì)細(xì)胞中去除抗原遞呈細(xì)胞可以降低肝細(xì)胞免疫源性,或者封閉抗原遞呈細(xì)胞的T細(xì)胞共刺激分子(B7蛋白)也可以調(diào)控免疫源性,這些都是解決免疫問題的措施。如何解決免疫排斥反應(yīng)是肝細(xì)胞移植應(yīng)用的重點(diǎn),但如何提供足夠數(shù)量、功能良好肝細(xì)胞更是決定肝細(xì)胞移植能否廣泛應(yīng)用的關(guān)鍵。相比全肝移植,肝細(xì)胞易于保存和運(yùn)輸,若能解決肝細(xì)胞的來源問題,肝細(xì)胞移植將來可在臨床上得到的廣泛應(yīng)用。 研究目的： 本實(shí)驗(yàn)通過向SD大鼠脾內(nèi)注射移植永生化HepGL肝細(xì)胞,對急性肝衰竭的大鼠模型進(jìn)行救治,研究永生化HepGL肝細(xì)胞在動(dòng)物體內(nèi)的功能,探討其是否具有替代并重建衰竭肝臟的能力。同時(shí)為其將來作為生物人工肝的種子細(xì)胞的應(yīng)用奠定理論基礎(chǔ)。 實(shí)驗(yàn)方法： 1.SD大鼠急性肝衰竭(Acute Hepatic Failure, AHF)模型的建立；采用90%肝部分切除術(shù)建立SD大鼠AHF模型,具體步驟如下： (1)術(shù)前準(zhǔn)備包括,術(shù)前SD大鼠禁食6h,改喂飲10%葡萄糖水；手術(shù)器械高壓滅菌；藥物(硫酸阿托品、乙醚、青霉素鈉等),耗材(5-0絲線、紗布、棉球、棉簽、一次性使用注射器等)等的準(zhǔn)備； (2)術(shù)前準(zhǔn)備完成后,將大鼠用乙醚麻醉,待大鼠麻醉后,將其固定于自制手術(shù)臺(tái)上,肌注阿托品0.03mmg(配成0.5ml鹽水溶液),并用50ml離心管內(nèi)放入浸濕了的無水乙醚的棉球,將管口對準(zhǔn)大鼠口鼻,持續(xù)吸入麻醉,麻醉深度的調(diào)節(jié)以大鼠停止躁動(dòng),呼吸平穩(wěn)為準(zhǔn)； (3)一切就緒后,備皮刀刮毛、腹部常規(guī)3次消毒、鋪無菌洞巾； (4)選擇腹部橫切口入腹,以剪刀剪開皮膚、肌肉及腹膜各層,以自制拉鉤充分暴露肝臟,解剖各肝葉間及與周圍組織之間的菲薄韌帶； (5)依次游離出各肝葉后,在右上葉(Right superior lobe,RSL)與肝中葉(Median lobe,ML)之間,游離出肝臟的Glisson系統(tǒng)頭支主干,以5-0絲線穿過結(jié)扎,可見肝中葉(ML)、左外葉(Left lateral lobe,LLL)迅速缺血,待肝葉變?yōu)橥咙S色后,5-0絲線結(jié)扎相應(yīng)肝葉的肝蒂后并切除；游離出右上葉(RSL)與右下葉(Right inferior lobe,RIL)的共同Glisson系統(tǒng)右支主干,5-0絲線結(jié)扎后同上依次切除右上葉與右下葉；僅保留包括前葉(Superior caudate lobe,SCL)與后葉(Inferior caudate lobe,ICL)的尾狀葉(Caudate lobe,CL)及腔靜脈旁部(paracaval liver,PL)(約占肝臟總量的10%)。檢查腹腔內(nèi)無出血后,于腹腔內(nèi)注射青霉素20萬單位溶液lml,依次縫合腹膜、肌層和皮膚,縫合皮膚前停止乙醚麻醉； (6)術(shù)后普通塊料喂養(yǎng),喂飲10%葡萄糖水,室溫18～22℃,光照12h/d。 2.永生化HepGL肝細(xì)胞移植； (1)實(shí)驗(yàn)分組： 實(shí)驗(yàn)組(n=21)—脾內(nèi)注射約2.5*107個(gè)永生化HepGL肝細(xì)胞(配成0.5mlDMEM混懸液)/只； 空白組(n=21)—未做任何治療的急性肝衰竭組； (2)實(shí)驗(yàn)組永生化HepGL肝細(xì)胞移植步驟： SD大鼠腹部行約1cm縱切口,用無菌棉簽伸入胃下提出脾臟,2.5*107個(gè)永生化HepGL肝細(xì)胞(配成0.5mlDMEM混懸液)大鼠脾內(nèi)注射,注射24h后SD大鼠行90%肝部分切除術(shù)。 3.觀察項(xiàng)目； (1)大體觀察：觀察空白組及實(shí)驗(yàn)組SD大鼠術(shù)后的飲食、精神狀態(tài)、活動(dòng)量及對外界刺激反應(yīng)及相關(guān)的癥狀表現(xiàn)等情況。 (2)存活率：記錄大鼠的死亡時(shí)間。 (3)生化檢查：收集Oh、24h、72h、7d、14d大鼠的靜脈血,檢測肝功能指標(biāo)包括：①谷丙轉(zhuǎn)氨酶(ALT)、②總膽紅素(Tbil)、③白蛋白(ALB)、④血氨(NH3)、⑤血糖(Glu)、⑥凝血時(shí)間(PT)等的變化。 (4)病理組織檢查：在24h、72h、7d、14d取大鼠肝臟及脾臟做組織病理切片及免疫組化,以了解大鼠肝臟急性肝衰竭病理變化、恢復(fù)情況以及移植的HepGL細(xì)胞在大鼠體內(nèi)的存活的情況。 實(shí)驗(yàn)結(jié)果： 1.大體觀察 實(shí)驗(yàn)組與空白組SD大鼠均在術(shù)后3-10min內(nèi)即可蘇醒翻身爬起,空白組大鼠12h后出現(xiàn)急性肝衰竭癥狀,包括精神萎靡,拒飲食,對外界反應(yīng)遲鈍,全身蜷縮,鼠毛豎立,尿色發(fā)黃、腹瀉,口鼻眼角出血等,大部分個(gè)體開始逐漸出現(xiàn)肝昏迷直至死亡。實(shí)驗(yàn)組大鼠12h后也有精神萎靡,食欲下降,對外界反應(yīng)遲鈍,全身蜷縮,鼠毛豎立等癥狀,但程度較輕,且無口鼻眼角出血等癥狀,也有部分個(gè)體出現(xiàn)肝昏迷并死亡,部分個(gè)體尸解可見腹腔內(nèi)有較清澈的腹水。 2.存活率 觀察兩組術(shù)后7d存活率,實(shí)驗(yàn)組7d存活率66.67%(14/21),死亡時(shí)間點(diǎn)分別為15h、35h、41h、45h、60h、62h;空白組7d存活率為28.57%(6/21),死亡時(shí)間點(diǎn)分別為16h、21h、23h、26h、32h、35h、38h、38h、41h、42h、50h、54h、60h、63h、70h,兩組死亡基本全部發(fā)生在3d內(nèi),大鼠存活超過3d,便可長期存活。 3.生化檢查 兩組大鼠術(shù)后各生化指標(biāo)均有明顯變化,兩組ALT、Tbil、NH3、PT等四項(xiàng)指標(biāo)均在術(shù)后24h上升到最高值,且四項(xiàng)指標(biāo)兩組之間均存在差異(P0.05),后逐漸恢復(fù)；兩組ALT、Tbil指標(biāo)72h也存在明顯差異(P0.05),在7d、14d點(diǎn)基本持平；兩組PT僅在24h有明顯差異；兩組ALB、Glu術(shù)后均出現(xiàn)下降,ALB指標(biāo)空白組在7d出現(xiàn)最低值,而實(shí)驗(yàn)組在72h出現(xiàn)最低值,兩組在24h、72h、7d各點(diǎn)均有明顯差異(P0.05); Glu指標(biāo)兩組均在24h出現(xiàn)最低值,在72h有明顯差異(P0.05)。 4.病理組織檢查 空白組大鼠術(shù)后24h時(shí)見殘余肝臟大體組織色澤淡黃,包膜腫脹,質(zhì)地松軟,觸之易碎,鏡下可見空泡變性和炎細(xì)胞浸潤；72h時(shí)殘余肝臟較前增大,邊緣可見血管增生,鏡下可見彌漫大量空泡變性和炎細(xì)胞浸潤,肝小葉結(jié)構(gòu)破壞。實(shí)驗(yàn)組大鼠注射后24h時(shí),脾臟紅腫,呈鮮紅色,術(shù)后肝臟病理表現(xiàn)同空白組,脾臟可見表面有斑點(diǎn)狀突起。免疫組化可見細(xì)胞在脾臟內(nèi)成團(tuán)聚集,在肝臟內(nèi)主要停留在肝血管竇部。 實(shí)驗(yàn)結(jié)論： 實(shí)驗(yàn)組大鼠生存質(zhì)量以及存活率明顯優(yōu)于空白組,兩組生化指標(biāo)在一定時(shí)間點(diǎn)存在明顯差異,組織病理發(fā)現(xiàn)實(shí)驗(yàn)組大鼠體內(nèi)存在移植的永生化HepGL肝細(xì)胞；說明永生化HepGL肝細(xì)胞在動(dòng)物體內(nèi)具備一定的生物學(xué)功能,能一定程度的替代部分肝功能,可作為生物人工肝的種子細(xì)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R657.3

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