本文選題：人臍帶間充質(zhì)干細(xì)胞 + 重癥急性胰腺炎��； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景和研究目的在重癥急性胰腺炎(severe acute pancreatitis,SAP)發(fā)病過(guò)程中,常累及多個(gè)臟器損傷,胰腺是首先受累的器官,常發(fā)生胰周液體積聚和胰腺組織的壞死,如果得不到及時(shí)治療,局部的炎性反應(yīng)將會(huì)擴(kuò)大,繼而可發(fā)展成全身炎癥反應(yīng)并累及遠(yuǎn)隔器官。腸道作為機(jī)體的天然屏障,具有阻隔腸道內(nèi)細(xì)菌和內(nèi)毒素等移位的屏障功能。當(dāng)腸屏障功能(intestinal barrier function,IBF)受到破壞時(shí),腸道內(nèi)細(xì)菌和內(nèi)毒素可發(fā)生移位,導(dǎo)致腸源性感染,繼而引發(fā)全身炎癥反應(yīng)綜合征(systemic inflammatory response syndrome,SIRS)以及多器官功能衰竭(multiple organ dysfunction syndrome,MODS),這也是重癥急性胰腺炎患者早期死亡的主要原因。針對(duì)SAP胰腺損傷及腸屏障功能障礙的治療,國(guó)際上尚無(wú)有效的治療方法,因此尋找一種行之有效的治療策略非常有必要。間充質(zhì)干細(xì)胞(mesenehymal stem cells,MSCs)是一類(lèi)具有多分化潛能的干細(xì)胞,研究發(fā)現(xiàn)其具有較強(qiáng)的免疫調(diào)節(jié)、抗炎及組織修復(fù)作用。已有研究表明骨髓來(lái)源的MSCs移植對(duì)SAP有很好的治療作用。然而,其應(yīng)用的種子細(xì)胞為骨髓MSCs,考慮到骨髓取材較困難,供體有限,且隨著年齡增長(zhǎng)骨髓MSC增殖能力、多向分化能力也會(huì)下降,這些因素限制了其在臨床上的進(jìn)一步應(yīng)用。相反,人臍帶間充質(zhì)干細(xì)胞(umbilical cord mesenchymal stem cells,uc MSCs)具有來(lái)源廣泛、取材方便、免疫源性弱、倫理限制少等優(yōu)點(diǎn),因此受到研究人員越來(lái)越多的關(guān)注。目前,國(guó)際上ucMSCs用于神經(jīng)系統(tǒng)疾病、血液體統(tǒng)疾病、糖尿病等疾病的治療當(dāng)中,然而其是否也對(duì)SAP具有治療作用卻鮮有報(bào)道。我們?cè)谇捌诘念A(yù)實(shí)驗(yàn)發(fā)現(xiàn)ucMSCs在SAP大鼠的胰腺及腸道組織中分布最多�；诖�,本實(shí)驗(yàn)擬探索ucMSCs對(duì)SAP大鼠胰腺及腸屏障損傷是否有保護(hù)作用,以期為人臍帶間充質(zhì)干細(xì)胞的臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。第一部分:人臍帶間充質(zhì)干細(xì)胞減輕重癥急性胰腺炎大鼠胰腺損傷的實(shí)驗(yàn)研究目的:觀察人臍帶間充質(zhì)干細(xì)胞對(duì)重癥急性胰腺炎大鼠胰腺組織損傷的作用方法:135只雄性SD(Sprague Dawley)大鼠隨機(jī)分成SHAM組、SAP組和SAP+ucMSCs組,各組分為12h、24h、72h三個(gè)時(shí)相點(diǎn),每個(gè)組45只大鼠,每個(gè)時(shí)相點(diǎn)15只大鼠。腹腔注射4%水合氯醛麻醉大鼠后,SHAM組開(kāi)腹,翻動(dòng)胰腺數(shù)次后關(guān)腹;SAP組采用微量注射泵以12ml/h的速度在胰管內(nèi)逆行注射5%�；悄懰徕c(0.1 ml/100 g)的方法制作SAP大鼠模型;SAP+ucMSCs組在SAP模型制作好后,通過(guò)尾靜脈注射CM-Di I標(biāo)記的ucMSCs(1×107個(gè)/kg)。各組于對(duì)應(yīng)的時(shí)相點(diǎn)處死動(dòng)物取材,其中72h時(shí)相點(diǎn)大鼠處死前統(tǒng)計(jì)72h大鼠的死亡率。熒光顯微鏡觀察ucMSCs在SAP大鼠胰腺歸巢情況,測(cè)定血清內(nèi)淀粉酶、脂肪酶、腫瘤壞死因子α(TNF-α)、白介素1β(IL-1β)、白介素4(IL-4)、白介素10(IL-10),HE染色觀察胰腺病理學(xué)改變并評(píng)分。結(jié)果:通過(guò)大鼠尾靜脈注射CM-Di I標(biāo)記的ucMSCs,在SAP+uc MSCs組各時(shí)相點(diǎn)的胰腺組織內(nèi)檢測(cè)到了CM-Di I陽(yáng)性細(xì)胞;與SAP組比較,SAP+ucMSCs組大鼠的死亡率降低,胰腺病理?yè)p傷減輕,血清淀粉酶及脂肪酶明顯降低,促炎因子(TNF-α、IL-1β)下降,抑炎因子(IL-4、IL-10)升高。第二部分:人臍帶間充質(zhì)干細(xì)胞減輕重癥急性胰腺炎大鼠腸屏障損傷的實(shí)驗(yàn)研究目的:觀察人臍帶間充質(zhì)干細(xì)胞對(duì)重癥急性胰腺炎大鼠腸屏障損傷的作用方法:45只雄性SD大鼠隨機(jī)分成SHAM組(n=15)、SAP組(n=15)和SAP+uc MSCs組(n=15),各組大鼠操作方法同第一部分。于術(shù)后24h處死動(dòng)物取材,熒光顯微鏡觀察uc MSCs在SAP大鼠腸道的歸巢情況,測(cè)定血清內(nèi)D-乳酸、內(nèi)毒素及小腸組織內(nèi)的TNF-α、IL-1β、角質(zhì)細(xì)胞生長(zhǎng)因子(keratinocyte growth factor,KGF),HE染色觀察小腸組織病理學(xué)改變并評(píng)分,胰腺、腸系膜淋巴結(jié)作細(xì)菌培養(yǎng)觀察細(xì)菌移位情況,透射電鏡觀察腸上皮細(xì)胞緊密連接、微絨毛情況,免疫熒光法及蛋白電泳法觀察小腸ZO-1、occludin蛋白的表達(dá)情況。結(jié)果:通過(guò)大鼠尾靜脈注射CM-Di I標(biāo)記的ucMSCs,在SAP+uc MSCs組大鼠腸道組織內(nèi)檢測(cè)到了CM-Di I陽(yáng)性細(xì)胞;與SAP組比較,SAP+ucMSCs組大鼠腸道內(nèi)的炎性因子TNF-?、IL-1?表達(dá)下降,KGF表達(dá)增加,血清內(nèi)的D-乳酸、內(nèi)毒素水平下降,腸系膜淋巴結(jié)和胰腺內(nèi)的細(xì)菌移位率降低,緊密連接破壞減輕,ZO-1、occludin蛋白表達(dá)增高。全文結(jié)論一、ucMSC移植可歸巢于SAP大鼠胰腺和腸道組織;二、ucMSCs移植不但顯著降低了SAP大鼠胰酶和脂肪酶的水平,減輕了SAP大鼠胰腺損傷,而且抑制了全身炎癥反應(yīng);三、uc MSCs移植明顯降低了SAP大鼠小腸炎性因子的水平,上調(diào)了小腸組織中KGF的表達(dá)量,改善了腸屏障功能。
[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緇，

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