【作者】 段征；

【导师】 汪维伟；

【作者基本信息】 重庆医科大学 ， 人体解剖与组织胚胎学， 2008， 硕士

【摘要】 目的骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs)具有高度增殖、自我更新能力,可向三个胚层的细胞分化[1],在机体组织损伤修复过程及细胞移植治疗等多个领域有广阔的应用前景。目前已有干细胞移植成功治疗炎症性肠病的报道[2],但其在消化道分布定位情况及治疗机制仍不明确。本实验拟①制作、比较大鼠溃疡性结肠炎(ulcerative colitis,UC)模型;②分离、培养、扩增、荧光标记大鼠BMSCs,观察其生物学特性;③观察标记的同种异体BMSCs经静脉移植后不同时间在UC及正常大鼠主要器官、消化道各段的分布情况:结肠内BMSCs是否明显多于消化道其余各段?溃疡病灶处BMSCs是否明显多于非溃疡部位?移植BMSCs后是否结肠病变修复优于非移植组?EGF能否促进BMSCs向溃疡部位迁移?为BMSCs在消化管组织工程以及肠黏膜损伤性疾病治疗研究中的应用,提供基础理论和实验依据。第一部分溃疡性结肠炎大鼠模型的建立与比较1材料与方法SD大鼠64只,随机分为:Ⅰ组(免疫+TNBS/乙醇)和Ⅱ组(TNBS/乙醇)各24只,Ⅲ组(乙醇)12只,Ⅳ组(生理盐水)4只。参照文献制作模型,于造模后第1、21天,第8周、12周处死大鼠,剖取全部结肠及回肠末段观察:①大体形态及结肠黏膜损伤情况;②光镜下结肠黏膜损伤;③免疫组化检测不同模型组肠壁组织表达TNF-α和IL-10的情况。2结果2.1肉眼及光镜显示:Ⅰ、Ⅱ组均出现糜烂、溃疡等典型UC结肠病变,Ⅰ组还伴有末段回肠病变;Ⅰ组的病变可维持8周,Ⅱ组病变3周后趋向愈合。Ⅲ组病变仅黏膜充血水肿和少量糜烂或浅表溃疡,第21天痊愈。Ⅳ组未见组织损伤。2.2免疫组织化学染色结果经图象分析显示:成模期内Ⅰ、Ⅱ、Ⅲ组结肠黏膜TNF-α表达高于正常对照的Ⅳ组,IL-10低于正常对照的Ⅳ组,差异均有统计学意义(P<0.01)。第二部分骨髓间充质干细胞的生物学特性与标记1材料与方法1.1取4-6周SD大鼠的股骨、胫骨骨髓,分离、培养、扩增获取BMSCs。检测BMSCs的贴壁率、生长曲线;流式细胞仪检测第3和第5代(P3和P5)BMSCs表达CD44、CD45和CD90的情况,以及细胞周期分析。1.2 Hoechst33342 (HCT)荧光标记P3-BMSCs后,不同时点荧光显微镜观察计算标记率。常规冻存复苏BMSCs,观察其生长情况。2结果2.1分离、培养的原代细胞易于贴壁,平均活细胞率为95.6±1.4%。培养3~12d,细胞数量迅速增多,具有典型的长梭状的形态及漩涡状排列的特征。传代BMSCs的生长快于原代细胞;P3～P5 BMSCs的生长滞留期约为24~48h,对数增殖期约为3~6d,7~8d后进入平台期;在含10%FBS的DMEM-F12培养基中每传代一次,细胞增加约2.2倍;6代后细胞出现形态异常的衰老征象。冻存的BMSCs复苏后生长较慢,复苏传代后可恢复至未冻存前传代细胞的生长状态。2.2流式细胞仪检测P3和P5细胞表面标记物CD44、CD90为阳性,CD45为阴性。CD44、CD90的均一性分别为98.26±0.48%及98.68±0.32%。细胞周期检测G0/G1期细胞约为90.81%,S+M+G2期为9.12%。2.3 HCT荧光标记后的BMSCs生长正常,标记率随时间延长缓慢减弱,4周后仍有约90% BMSCs可观察到荧光。第三部分骨髓间充质干细胞移植修复溃疡性结肠炎的作用1材料与方法1.1 SD大鼠60只随机分为A、B、C和D组,每组15只。A、B、C组以免疫+TNBS/乙醇复合法建立UC模型。制备HCT荧光标记的P3～P5 BMSCs悬液备用。模型制备完成后24 h内,4组大鼠均经尾静脉注射1ml不同液体:A组为生理盐水的移植对照组;B组为荧光标记的BMSCs悬液(1×106)的移植组;C组为含300μg EGF的荧光标记BMSCs悬液(1×106)的移植组;D组为荧光标记的BMSCs悬液(1×106)的模型对照组。1.2移植后第3、7、14天各处死5只,解剖观察结肠病变。取B、C和D组的骨髓、肺、肝、脾和消化道组织行冰冻切片,荧光显微镜下观察荧光标记的BMSCs在骨髓、肺、肝、脾、食道、胃、小肠和结肠内的分布。并检测各组各时间点切片随机10个20倍物镜视野上的荧光积分光密度(integrated optical density, IOD)值,观察其在各器官、组织间的数量差异。1.3取A、B、C和D组的结肠制成HE染色石蜡切片,观察结肠组织的光镜下的溃疡病变。另取结肠石蜡切片经免疫组化染色后观察TNF-α和IL-10的表达。2结果2.1 HCT标记细胞在肺、肝、脾、骨髓分布有一定时间规律性:肺、肝、脾的荧光IOD值7d>3d>14d,骨髓的荧光IOD 7d >14d >3d;第3、7d肺的荧光IOD值和第14d时骨髓的荧光IOD值略高于其他器官;同一器官同一时间点各组荧光IOD值的差异无统计学意义。2.2移植第3、7、14d,B、C和D组食道、胃及小肠的黏膜层均可见标记的BMSCs,同一器官同一时间点各组荧光IOD值的差异无统计学意义。2.3移植第3d,各组结肠与同组食管、胃及小肠荧光IOD值的差异无统计学意义;移植第7、14d,B、C组结肠的荧光IOD值高于同组食道、胃及小肠(P< 0.05);移植14d,B、C组结肠的荧光IOD值高于D组(P<0.05),B和C组间结肠的荧光IOD值的差异无统计学意义。2.4镜下观察,标记的BMSCs主要见于各移植组各时间点的结肠黏膜层,偶见于黏膜下层及肌层。第14d,B、C组结肠病变部位的BMSCs最多,黏膜糜烂和溃疡坏死病灶均可见较多BMSCs,在溃疡周边黏膜层略多于非病变部位结肠黏膜。2.5 A、B和C组结肠炎症及溃疡表现为全结肠性,可见黏膜充血、水肿、上皮细胞坏死脱落、大量炎症细胞浸润及溃疡。D组结肠无异常。移植后第14d,B和C组大鼠结肠黏膜损伤修复程度明显优于A组,B和C组大鼠结肠黏膜损伤修复程度无明显差异。2.6移植后第3、7、14d ,A、B、C组结肠黏膜TNF-α表达高于D组,IL-10低于D组,差异均有统计学意义(P<0.01);第14d, B、C组TNF-α表达低于A组,而IL-10高于A组,差异均有统计学意义(P<0.01);B、C组TNF-α和IL-10表达的差异无统计学意义。全文结论1. 3种UC大鼠模型中,以免疫复合法所建模型为较理想的UC动物模型。2.用贴壁筛选法,成功分离得到大鼠BMSCs,流式细胞仪分析培养P3、P5 BMSCs具有MSCs的表面标记特征,也具有干细胞的细胞周期特点;传代BMSCs的生长较原代细胞快,在含10%FBS的DMEM-F12培养基中每传代一次,细胞增加约2.2倍;冻存对BMSCs的生长无明显影响;P6 BMSCs出现衰老征象。3. Hoechst33342荧光标记BMSCs是短期示踪细胞的有效手段。4.移植的BMSCs在体内广泛分布,在7d的肺14d的骨髓中含量略高于肝和脾,提示移植14d时,多数干细胞仍在血循环中。5.移植的BMSCs 3d即可迁移至结肠,多定居于黏膜层。移植后14d病变结肠BMSCs较其余消化道、以及正常结肠多,病变结肠黏膜层BMSCs较正常结肠黏膜多,提示BMSCs可向损伤组织定向迁移。6.移植BMSCs后14d,移植组结肠黏膜损伤修复程度明显优于非移植组,提示BMSCs移植可修复或促进损伤结肠组织修复。7. BMSCs移植UC大鼠IL-10表达增高而TNF-α表达降低,表明移植的BMSCs可能通过调节肠道TNF-α、IL-10等炎症细胞因子达到免疫平衡来修复UC结肠病灶。8. BMSCs移植组与BMSCs+EGF联合移植组的BMSCs在消化道各段以及结肠病变部位或周边的分布、炎症因子的表达上均无明显差异,提示单次注射EGF无促进BMSC向损伤部位迁移及损伤修复的作用。更多还原

【Abstract】 Objective: Bone marrow mesenchymal stem cells(BMSCs) are considered to maintain the high proliferative capacity, have the potential to differentiate into all cells in three germinal layers, and is useful in the fields of tissue engineering, wound repair and cell transplant therapy. At present, it had been reported that transplantation of BMSCs can be the new effective therapy for inflammatory bowel disease, nevertheless, the theory about distribution of transplanted BMSCs and treatment mechanism in colon is not clear. This study try to compare several UC models to find the most proper one; to isolate, culture, amplificate, mark the rat BMSCs with fluorescence, and observe the bionomics of BMSCs; to observe the distribution of the marked BMSCs in different organs and tissues at different time points after transplantation of BMSCs in UC model of rats. Whether or not the number of BMSCs in colon is more than in other segments of digestive canal? the number of BMSCs in ulcerative focus of infection of colon is more than in non-ulcerative places? Whether or not the repair of injured tissue in UC models after transplanting is better than without transplanting? Whether or not EGF can encourage BMSCs moving to ulcerative focus of infection? Try to provide the basic theory and experiment for the future research of BMSCs in the tissue engineering and treating intestinal mucosa injury disease.PARTⅠTHE ESTABLISHMENT AND COMPARING OF ULCERATIVE COLITIS MODEL IN RATS1. Materials and Methods1.1 SD rats were randomly distributed into groupⅠ(immune- combined TNBS/ethanol), groupⅡ(TNBS/ethanol), groupⅢ(50% ethanol) and groupⅣ(normal saline). The model were made according to literatures, rats were sacrificed at day 1, 21 and week 8, 12 after enema with different material in 4 groups to observe the macroscopical focus of infection in bowels, the microscopical changes and the expressing-level of TNF-αand IL-10 in mucous membrane of colon.2. Results2.1 The observation with naked eye and light microscope show that the typical erosion and ulcer changes appeared in colon of groupⅠandⅡ, and there was accompanying pathological changes in the end piece of ileum in groupⅠ. The pathological changes for groupⅠcould keep for 8 weeks, while those of groupⅡtrended to heal 3 weeks later. The pathological changes in groupⅢonly were hyperemia, oedema and a few anabrosis or superficial ulcer, keeped for 21days. There was no tissue damage in colon of groupⅣ. 2.2 Immunohistochemistry revealed: the level of TNF-αin mucous membrane of colon of groupⅠ,Ⅱ,Ⅲwere much higher than that of groupⅣ, but the expression of IL-10 were lower. The difference of expression of TNF-αand IL-10 was statistical significance between groupⅠ,Ⅱ,Ⅲand groupⅣ(P<0.01).PARTⅡTHE BIOLOGIC CHARACTER AND LABELLING OF BMSC1. Materials and Methods1.1 Obtain limbs bone marrow samples from SD rats aged 4 to 6 weeks. BMSCs had been isolated, cultured and proliferated in vitro. The adherence rate, growth curve of BMSCs had been observed. The express of CD44, CD45, CD90 and status of cell cycle in the 3th and 5th generation of BMSCs ( P3 and P5 ) are detected by flow cytometry.1.2 After Hoechst33342 (HCT)f luorescent labelling P3-BMSCs, the marking condition in different times were observed by fluorescence microscope, and the marking rates were calculated. The growth information of BMSCs after freezed and resusing in common practice has been observed.2. Results2.1 The primary cells can adherence easily,the average rate of living cell is 95.6±1.4%. The BMSCs proliferated rapidly from cultured 3 to 12 days, have typical feature of spindle-shape and whirlpool arrangement in culture. The subcultured cells proliferated faster than primary cells. The detention period of BMSCs is 24~48h, the log period is 3~6d, the platform period is 7~8d. The BMSCs can proliferate more than 2.2 times in DMEM-F12 (contain 10%FBS). The primary revivaled BMSCs grew slowly, and after subcultured, the cells restored growth condition as before freezing. After 6 generation, BMSCs appear the signs of senility.2.2 The result of flow cytometer–detection showed that the BMSCs in generation 3 and 5 express CD44, CD90, but not express CD45. Cell cycle status analysis revealed that a small population of the cells were actively engaged in proliferation (S+G2+M=9.12%), and more than 90.81% of cells were in G0/G1 phase.2.3 The BMSCs marked with HCT proliferated normaly, the marking rate of the cells decreased slowly, which could be about 90% after the fourth week.PARTⅢTRANSPLANT AND REPAIR EFFECT OF BMSCS IN RAT ULCERATIVE COLITIS MODEL1. Materials and Methods1.1 SD rats were randomly distributed into 4 groups, Colitis was induced in group A, B and C with immune- combined TNBS/ethanol. Following the induction of colitis, each rat in 4 groups received caudal vein injection of 1 ml different fluids, which were: normal sodium for group A, HCT marked BMSCs(1×106)for group B and D, HCT marked BMSCs (1×106)with 300μg for group C.1.2 5 Rats were sacrificed at day 3, 7 and 14 after injection in each group, dissected and observed the colonic pathological changes. The bone marrow, lung, spleen, liver, esophagus, stomach, small intestine and colon of group B, C and D were prepared as frozen sections to observe the distribution of HCT marked BMSCs in these organs with fluorescent microscope. The value of integrated optical density (IOD) in each group and different time points were detect in 10 fields of vision of 20 times object lens,to survey the quantitive discrepancy of HCT marked BMSCs in these organs.1.3 The colon in groups A, B, C and D were prepared as paraffin section stained by HE to observe the microscopical pathological-changes. Another parts paraffin sections were stained by immuno histochemistry method to observe the express of TNF-αand IL-10 in colon.2. Results2.1 There was a regular time pattern about IOD level of BMSCs in bone marrow, lung, spleen and liver: IOD level of BMSCs in lung, spleen and live was 7d > 3d > 14d;the IOD level of BMSCs in bone marrow was 7d > 14d > 3d. At day 7, the highest IOD of BMSCs was in lung in all organs, and at day 14, the highest one was in bone marrow(P<0.05). The difference of the IOD level in same organ at same time point in all groups did not showed the statistically significant.2.2 On the 3, 7 and 14d, HCT marked BMSCs in groups B, C and D were seen in mucosa layer of esophagus, stomach, ileum. There was no statistically significant among the difference in the IOD level of esophagus, stomach, ileum at same time point in all groups.2.3 On the 3 day of transplantation, the difference of the IOD level between colon and esophagus, stomach, ileum in same groups did not showed the statistically significant. But, on the 7, 14 day of transplantation, the IOD level in group B and C were higher than that in group D(P<0.05). The difference of the IOD level between group B and C had no statistically significant.2.4 Under light microscope, the HCT marked BMSCs were seen mainly in colonic mucosa of group B, C and D in each time point, occasionally were seen in submucosa and tunica muscularis. On the 14 day of transplantation, the marked BMSCs in diseased region of colon was maximum, more number of marked BMSCs was appeared in the mucosa anabrotic focus of infection, and those in the peripheric mucosa of ulcer was exceed than that in colonic mucosa of non-anabrotic focus of infection.2.5 There was macroscopic and microscopic evidence of colonic mucosa congestion, edema, epithelial necrosis, inflammatory cell infiltration and ulcer in group A, B and C, but no one be noted in group D. The extent of reparation of colonic mucosa-injury in group B and C were surpassed obviously than that in group A at day 14, but there was no difference between group B and C.2.6 At day 3, 7, 14 of transplantation, the expression of TNF-αin mucous membrane of colon in group A, B, C were higher than that in group D, but the expression of IL-10 lower than that in group A(P<0.01) . At day 14, the expression-level of TNF-αin colonic mucosa in group B, C were lower than that of group A, but the expression of IL-10 were opposite (P<0.01). The difference of expression of TNF-αand IL-10 was no statistically significant.Conclusion of whole article1. Immune-combind TNBS/ethanol method was the ideal method to establish the UC model comparatively.2. The rats BMSCs have been isolated successfully by adherence screening. The result of Flow cytometer analyzed had shown that the cells of passage 3, 5 possess the character of surface antigens of MSCs and the cell cycle feature of stem cells. The subcultured cells proliferated faster than primary cells; The BMSCs could proliferate more than 2.2 times in DMEM-F12 (contain 10% FBS). The grow of BMSCs was not effected by freezing.After 6 generation, BMSCs appear the signs of senility.3. HCT marked cells was a useful method for tracing BMSCs in short period.4. Transplanted BMSCs widely distributed throught body. BMSCs in lung at day3, 7 and in bone marrow at day 14 were higher than that in liver and spleen. It hinted that most BMSCs retained in blood circulation.5. Transplanted BMSCs could migrate and distribute in mucosa of colon on day 3. The number of BMSCs in injured colon was more than that in esophagus, stomach, ileum and nomal colon. The number of marked BMSCs in the mucosa of anabrotic focus of infection was more than that in normal colonic mucosa. It indicated that BMSCs may trended to migrant into injured tissue.6. The extent of reparation of colonic mucosa-injury in BMSCs transplanting group was surpassed obviously than that in non- BMSCs transplanting group. It show us that transplantation of BMSCs could repair damage or promote repairing damage in colon.7. The higer expression of IL-10 and the lower expression of TNF-αin colinic mucosa of BMSCs transplanted UC rats indicated that BMSCs migrated in colonic mucosa of UC rat might be regulate the inflammatory cell factors, TNF-αand IL-10, to achieve immunologic balance, thus,promote the recovery of colonic focus of infection in UC rat.8. Between BMSCs transplantation groups with EGF or without EGF, there was no difference in the number and the distribution of BMSCs in gut or injured colon and the expression of inflammation factors. It indicated that injection of EGF once could not encourage BMSCs migranting to the injured tissue or repairing the damage of colonic tissue.更多还原