人脐带间充质干细胞治疗重症急性胰腺炎的实验研究

发布时间：2018-04-15 12:50

  本文选题：人脐带间充质干细胞 + 重症急性胰腺炎　； 参考：《第三军医大学》2017年硕士论文

【摘要】：研究背景和研究目的在重症急性胰腺炎(severe acute pancreatitis,SAP)发病过程中,常累及多个脏器损伤,胰腺是首先受累的器官,常发生胰周液体积聚和胰腺组织的坏死,如果得不到及时治疗,局部的炎性反应将会扩大,继而可发展成全身炎症反应并累及远隔器官。肠道作为机体的天然屏障,具有阻隔肠道内细菌和内毒素等移位的屏障功能。当肠屏障功能(intestinal barrier function,IBF)受到破坏时,肠道内细菌和内毒素可发生移位,导致肠源性感染,继而引发全身炎症反应综合征(systemic inflammatory response syndrome,SIRS)以及多器官功能衰竭(multiple organ dysfunction syndrome,MODS),这也是重症急性胰腺炎患者早期死亡的主要原因。针对SAP胰腺损伤及肠屏障功能障碍的治疗,国际上尚无有效的治疗方法,因此寻找一种行之有效的治疗策略非常有必要。间充质干细胞(mesenehymal stem cells,MSCs)是一类具有多分化潜能的干细胞,研究发现其具有较强的免疫调节、抗炎及组织修复作用。已有研究表明骨髓来源的MSCs移植对SAP有很好的治疗作用。然而,其应用的种子细胞为骨髓MSCs,考虑到骨髓取材较困难,供体有限,且随着年龄增长骨髓MSC增殖能力、多向分化能力也会下降,这些因素限制了其在临床上的进一步应用。相反,人脐带间充质干细胞(umbilical cord mesenchymal stem cells,uc MSCs)具有来源广泛、取材方便、免疫源性弱、伦理限制少等优点,因此受到研究人员越来越多的关注。目前,国际上ucMSCs用于神经系统疾病、血液体统疾病、糖尿病等疾病的治疗当中,然而其是否也对SAP具有治疗作用却鲜有报道。我们在前期的预实验发现ucMSCs在SAP大鼠的胰腺及肠道组织中分布最多。基于此,本实验拟探索ucMSCs对SAP大鼠胰腺及肠屏障损伤是否有保护作用,以期为人脐带间充质干细胞的临床应用提供实验依据。第一部分:人脐带间充质干细胞减轻重症急性胰腺炎大鼠胰腺损伤的实验研究目的:观察人脐带间充质干细胞对重症急性胰腺炎大鼠胰腺组织损伤的作用方法:135只雄性SD(Sprague Dawley)大鼠随机分成SHAM组、SAP组和SAP+ucMSCs组,各组分为12h、24h、72h三个时相点,每个组45只大鼠,每个时相点15只大鼠。腹腔注射4%水合氯醛麻醉大鼠后,SHAM组开腹,翻动胰腺数次后关腹;SAP组采用微量注射泵以12ml/h的速度在胰管内逆行注射5%牛磺胆酸钠(0.1 ml/100 g)的方法制作SAP大鼠模型;SAP+ucMSCs组在SAP模型制作好后,通过尾静脉注射CM-Di I标记的ucMSCs(1×107个/kg)。各组于对应的时相点处死动物取材,其中72h时相点大鼠处死前统计72h大鼠的死亡率。荧光显微镜观察ucMSCs在SAP大鼠胰腺归巢情况,测定血清内淀粉酶、脂肪酶、肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)、白介素4(IL-4)、白介素10(IL-10),HE染色观察胰腺病理学改变并评分。结果:通过大鼠尾静脉注射CM-Di I标记的ucMSCs,在SAP+uc MSCs组各时相点的胰腺组织内检测到了CM-Di I阳性细胞;与SAP组比较,SAP+ucMSCs组大鼠的死亡率降低,胰腺病理损伤减轻,血清淀粉酶及脂肪酶明显降低,促炎因子(TNF-α、IL-1β)下降,抑炎因子(IL-4、IL-10)升高。第二部分:人脐带间充质干细胞减轻重症急性胰腺炎大鼠肠屏障损伤的实验研究目的:观察人脐带间充质干细胞对重症急性胰腺炎大鼠肠屏障损伤的作用方法:45只雄性SD大鼠随机分成SHAM组(n=15)、SAP组(n=15)和SAP+uc MSCs组(n=15),各组大鼠操作方法同第一部分。于术后24h处死动物取材,荧光显微镜观察uc MSCs在SAP大鼠肠道的归巢情况,测定血清内D-乳酸、内毒素及小肠组织内的TNF-α、IL-1β、角质细胞生长因子(keratinocyte growth factor,KGF),HE染色观察小肠组织病理学改变并评分,胰腺、肠系膜淋巴结作细菌培养观察细菌移位情况,透射电镜观察肠上皮细胞紧密连接、微绒毛情况,免疫荧光法及蛋白电泳法观察小肠ZO-1、occludin蛋白的表达情况。结果:通过大鼠尾静脉注射CM-Di I标记的ucMSCs,在SAP+uc MSCs组大鼠肠道组织内检测到了CM-Di I阳性细胞;与SAP组比较,SAP+ucMSCs组大鼠肠道内的炎性因子TNF-?、IL-1?表达下降,KGF表达增加,血清内的D-乳酸、内毒素水平下降,肠系膜淋巴结和胰腺内的细菌移位率降低,紧密连接破坏减轻,ZO-1、occludin蛋白表达增高。全文结论一、ucMSC移植可归巢于SAP大鼠胰腺和肠道组织;二、ucMSCs移植不但显著降低了SAP大鼠胰酶和脂肪酶的水平,减轻了SAP大鼠胰腺损伤,而且抑制了全身炎症反应;三、uc MSCs移植明显降低了SAP大鼠小肠炎性因子的水平,上调了小肠组织中KGF的表达量,改善了肠屏障功能。
[Abstract]:Study background and purpose of the study on severe acute pancreatitis (severe acute, pancreatitis, SAP) in the pathogenesis, often involving multiple organ injury, pancreas is the first organ, often peripancreatic fluid collections and pancreatic tissue necrosis, if not treated in time, will expand the local inflammatory reaction, then can the development of a systemic inflammatory response involving distant organs. The gut as a natural barrier of the body, can prevent intestinal bacteria and endotoxin translocation. When the barrier function of the intestinal barrier function (intestinal barrier, function, IBF) damage, intestinal bacteria and endotoxin translocation, leading to intestinal infection, leading to the body inflammatory reaction syndrome (systemic inflammatory response syndrome, SIRS) and multiple organ failure (MODS multiple organ dysfunction syndrome), which is suffering from severe acute pancreatitis The main causes of early death. SAP treatment for pancreatic injury and intestinal barrier dysfunction, there is no effective treatment, so look for an effective treatment strategy is very necessary. Mesenchymal stem cells (mesenehymal stem cells, MSCs) is a class of multiple differentiation potential stem cells, the study found it has strong immunomodulatory, anti-inflammatory and tissue repair. Studies have shown that bone marrow derived MSCs transplantation has good effects on treating SAP. However, the application of seed cells for bone marrow MSCs, taking into account the bone marrow were more difficult, the donor is limited, with the age growth of bone marrow MSC proliferation, multilineage differentiation ability fall, these factors limit its further application in clinic. On the contrary, human umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells, UC MSCs) has broad source, convenient , weak immunogenicity, ethical advantages of fewer restrictions, so researchers have more and more attention. At present, the international ucMSCs for diseases of the nervous system, blood system diseases, treatment of diabetes and other diseases, but also on whether SAP has therapeutic effects are rarely reported. We found that in the pre experimental pre ucMSCs up located in the pancreas and intestinal tissue of SAP rats. Based on this, to explore whether ucMSCs has protective effects on pancreas and intestinal barrier injury to rat SAP this experiment, in order to provide experimental basis for clinical application of human umbilical cord mesenchymal stem cells. The first part: experimental study of human umbilical cord mesenchymal stem cells reduce pancreatic injury in rats with severe acute pancreatitis: Observation of human umbilical cord mesenchymal stem cells on the function of pancreatic tissue of rats with severe acute pancreatitis injury: 135 male SD (Sprague Dawley) rats were randomly divided into 鎴怱HAM缁，

本文编号：1754182