人脐血源基质细胞对造血损伤模型小鼠体内造血调控作用的研究

发布时间：2018-06-25 11:10

本文选题：人脐血源基质细胞 + 人脐血源间充质干细胞　；参考：《泸州医学院》2014年硕士论文

【摘要】：目的：本课题采用密度梯度离心收集获得人脐血单个核细胞（mononuclear cells, MNCs），经过传代培养得到人脐血源基质细胞（humanumbilical cord blood-derived stromal cells, hUCBDSCs）、人脐血源间充质干细胞（human umbilical cord blood-derived mesenchymal stem cells,hUCBDMSCs）；观察两类细胞形态结构、生长增殖特点，流式细胞仪技术测得两类细胞的表面抗原；经DiI标记后分别输注入造血功能损伤模型小鼠模型体内，动态检测小鼠静脉血红细胞、白细胞、血小板、血红蛋白浓度，了解外周血血象恢复情况；动态检测两类细胞对骨髓造血微环境的修复情况，深入比较人脐血源基质细胞与其间充质干细胞恢复造血的能力，达到确立在临床可使用人脐血源基质细胞即可恢复造血这一理论体系提供了动物实验依据，解决了临床上因使用促造血生长因子带来的副作用及产生昂贵费用这一实际问题；也给长期探索早期造血恢复方法的血液学工作者带来了新的思路。方法：1.细胞分离与培养：采用密度梯度离心收集获得脐血单个核细胞，用DMEM/F-12、MSCM培养体系分别进行单个核细胞的体外培养，得到纯度较高的hUCBDSCs及hUCBDMSCs，倒置显微镜下动态观察两类细胞生长增殖情况及形态特点，利用流式细胞技术检测两类细胞表面抗原；2.DiI标记两类细胞进行示踪；3.建立小鼠造血损伤模型：X射线辐照BALB/c小鼠（总剂量5Gy，吸收率100cGy/min，时间5min，距离100cm）；4.实验分组：将造型损伤模型小鼠随机分为3组：hUCBDSCs组、hUCBDMSCs组、对照组；5.分别经小鼠尾静脉移植注入hUCBDSCs（1.0×106/只）、hUCBDMSCs（1.0×106/只）、生理盐水（0.2ml），动态记录三组小鼠的精神状态、饮食情况等一般生理状态及生存状况，计算各组生存率；分别于3d、7d、14d取静脉血检测红细胞、白细胞、血小板、血红蛋白，了解外周血血象恢复情况；3d、7d、14d分别进行骨髓涂片观察两类细胞在小鼠骨髓归巢情况、骨髓活检了解两类细胞对骨髓造血微环境损伤的修复作用；6.统计学分析：应用SPSS22.0统计分析软件对所获得的数据进行处理，计量数据以均数±标准差(ˉx±s)表示，各组间采用单因素方差分析，两组间数据比较应用t检验，检验水准α设定为0.05，P0.05差异有统计学意义；计数资料采用率描述。结果：1.采用密度梯度离心可获得单个核细胞，经体外传代培养得到纯度较高的hUCBDMSCs及hUCBDSCs；MSCM培养体系培养原代细胞，24h后细胞呈不规则形、类圆形，分布均匀，胞体小，透明；48h后细胞贴壁，数量增多，部分细胞开始向两极伸展；7-12d细胞以梭形为主，集落样分布，细胞内颗粒少，透明，细胞间界限清楚；14-16d可铺满瓶底；传代后细胞形态一致，生长增殖较原代快，经1-2d潜伏期后进入对数生长期，呈均匀细长梭形，6-7d后进入平台期。F-12培养体系培养原代细胞，24h后细胞呈类圆形、不规则形，分布均匀，胞质透明；48h后细胞数量增多，贴壁生长；7-12d细胞集落样分布，形态多样，以类圆形、扁形为主，细胞间界限清楚，胞质透明，颗粒少；传代后细胞生长较原代快，经1-2d潜伏期后进入对数生长期，呈圆形或扁形，6d后进入平台期。两类细胞生长曲线变化趋势一致：传代后1-2d细胞处于潜伏期，生长增殖缓慢；48h后细胞指数增长，增殖、活力旺盛，进入对数生长期；6-7d后细胞生长增殖明显减缓，由对数生长期进入平台期。hUCBDSCs对数生长期前期增殖速度较hUCBDMSCs缓慢，后期增长速度快于hUCBDMSCs。2.流式细胞术检测细胞表面抗原的表达，hUCBDMSCs表达CD29、CD44、CD106、STRO-1，不表达CD34、CD45；hUCBDSCs表达CD44、CD45、Lm、Fn，不表达CD29、STRO-1，两类细胞表面抗原的表达与大量研究结果一致，证实DMEM/F-12培养体系可培养出hUCBDSCs，MSCM培养体系可培养出hUCBDMSCs。3.模型的建立：非致死剂量辐照BALB/c小鼠（剂量5Gy，能量6MeV，距离100cm，，吸收率100cGy/min，时间5min），可造成小鼠急性造血功能损伤。4.DiI标记第三代hUCBDMSCs、hUCBDSCs，两类细胞在激光共聚焦显微镜下显橙红色荧光；形态与普通倒置相差显微镜一致；两类细胞标记率均为100%。5.血象的恢复情况：动态监测血常规，结果显示移植后3d，三组小鼠白细胞、血小板、红细胞计数及血红蛋白浓度均无显著差异(P0.05)；移植后第7d，hUCBDSCs组及hUCBDMSCs组白细胞、血小板、红细胞、血红蛋白恢复情况均显著高于对照组(P0.05)，且hUCBDSCs组白细胞、血小板、红细胞均显著高于hUCBDMSCs组(P0.05)，血红蛋白无浓度明显差异(P0.05)。移植后14d，hUCBDSCs组及hUCBDMSCs组白细胞、血小板、红细胞、血红蛋白恢复均显著高于对照组(P0.05)，且hUCBDSCs组白细胞、血小板计数及血红蛋白浓度显著高于hUCBDMSCs组(P0.05)，红细胞计数无明显差异(P0.05)。6.两类细胞的归巢：动态监测骨髓示踪细胞，结果显示两类细胞均可在移植后期稳定定植于骨髓，且hUCBDSCs组较hUCBDMSCs组先归巢于骨髓(P0.05)。7.骨髓病理切片：移植后3d三组小鼠骨髓均出现造血功能抑制，有核细胞增生不良，非造血细胞比例增加；移植后7d三组小鼠造血功较前均有恢复，有核细胞成簇分布，hUCBDSCs组、hUCBDMSCs组骨髓增生均优于对照组，且hUCBDSCs组优于hUCBDMSCs组；移植后14d对照组骨髓造血功能恢复情况较前无明显改变，hUCBDSCs组与hUCBDMSCs组造血功能明显恢复，有核细胞增生活跃，人脐血源基质细胞组可见大量巨核细胞。提示脐血干细胞移植可作为修复放/化疗后造血微环境损伤的一种安全有效方法，具有潜在的临床应用前景。结论：1.利用密度梯度离心，体外培养可获得高纯度的hUCBDMSCs及hUCBDSCs。2.hUCBDSCs、hUCBDMSCs移植入小鼠体内后，可明显促进白细胞、血小板、红细胞恢复，且hUCBDSCs恢复血象作用更强，骨髓抑制程度更轻。3.hUCBDSCs、hUCBDMSCs移植入造血损伤模型小鼠体内后，均可稳定定植于骨髓，且hUCBDSCs较hUCBDMSCs先归巢于骨髓；两类细胞对骨髓造血功能恢复均有明显促进作用，且hUCBDSCs优于hUCBDMSCs。
[Abstract]:Objective : To obtain human umbilical cord blood - derived stromal cells ( hUCBDSCs ) and human umbilical cord blood - derived mesenchymal stem cells ( hUCBDMSCs ) by means of density gradient centrifugation .
Two kinds of cell morphology , growth and proliferation were observed , and the surface antigens of two kinds of cells were measured by flow cytometry .
After DiI labeling , the blood cells , white blood cells , blood platelet and hemoglobin concentration were measured in mice model mice respectively , and the recovery of peripheral blood image was understood .
dynamic detection of two types of cells to repair the hematopoietic microenvironment of the bone marrow , in - depth comparison of the ability of the human umbilical cord blood source stromal cells and the mesenchymal stem cells to recover the hematopoietic stem cells , thereby achieving the theoretical system for establishing the hematopoietic system which can recover the hematopoietic stem cells by using the human umbilical cord blood source stromal cells , and solves the practical problem of the side effects caused by the use of the hematopoietic growth factor and the expensive cost in clinic ;
Methods : 1 . Isolation and culture of cells : 1 . Isolation and culture of cells : Using density gradient centrifugation to collect mononuclear cells from umbilical cord blood , the cells were cultured in vitro by DMEM / F - 12 and MSCM culture system . Two kinds of cell surface antigens were detected by flow cytometry .
2 . DiI labeling two types of cells ;
3 . Establishment of mouse hematopoietic injury model : X - ray irradiation BALB / c mice ( total dose 5Gy , absorption rate 100cGy / min , time 5min , distance 100cm ) ;
4 . Experimental group : The model mice were randomly divided into three groups : hUCBDSCs group , hUCBDMSCs group and control group ;
5 . hUCBDSCs ( 1.0 脳 106 / rats ) , hUCBDMSCs ( 1.0 脳 106 / rats ) and physiological saline ( 0.2 ml ) were injected into the tail vein of mice respectively , the mental status , diet and survival status of three groups of mice were dynamically recorded , and the survival rates were calculated .
Blood samples of red blood cells , white blood cells , platelets and hemoglobin were taken from venous blood at 3d , 7d , 14d , respectively .
Bone marrow smear and bone marrow biopsy were used to observe the effects of two kinds of cells on the repair of bone marrow hematopoietic microenvironment .
6 . Statistical analysis : The data obtained were analyzed by SPSS 10.0 statistical analysis software , and the measured data was expressed by mean 卤 standard deviation ( 藟x 卤 s ) . Single - factor variance analysis was used among the groups , and t - test was applied between the two groups .
Results : 1 . Single core cells can be obtained by density gradient centrifugation , and hUCBDMSCs and hUCBDSCs with higher purity can be obtained through in vitro subculture .
MSCM culture system cultured primary cells , the cells were irregular , round , uniform , small and transparent after 24 h .
After 48h , the cells were adherent , the number of cells increased , some cells began to spread to the two poles ;
7 - 12d cells were spindle - shaped , colony - like distribution , few intracellular particles , clear and clear inter - cell boundaries ;
14 - 16d can be filled with a bottle bottom ;
After 1 - 2 days incubation period , the cells showed round shape , irregular shape , uniform distribution and transparent cytoplasm .
After 48h , the number of cells increased and the adherent cells grew ;
7 - 12d cell colonies were distributed in a variety of shapes , such as round shape , flat shape , clear cell lines , clear cytoplasm and few particles .
After 1 - 2 days incubation period , the cell growth was faster than that of the primary culture . After 1 - 2days incubation period , the cells were transformed into logarithmic growth phase , and then entered the plateau phase after 6 days . The two types of cell growth curves were consistent : 1 - 2 days after passage , the cells were in latency and the growth was slow ;
After 48h , the cell index increased , the proliferation and vitality were vigorous , and the logarithmic growth period was entered .
The proliferation rate of hUCBDSCs was slower than that of hUCBDMSCs , and the growth rate of hUCBDMSCs was faster than that of hUCBDMSCs .
hUCBDSCs were expressed by hUCBDSCs , and the expression of CD29 , STRO - 1 , two kinds of cell surface antigens was consistent with the results of a large number of research results . It was confirmed that the culture system of DMEM / F - 12 could be cultured for hUCBDSCs .
the morphology is consistent with the common inverted phase contrast microscope ;
The results showed that there was no significant difference in leukocyte , platelet , erythrocyte count and hemoglobin concentration in three groups ( P0.05 ) .
The WBC , platelet count and hemoglobin concentration in hUCBDSCs and hUCBDMSCs were significantly higher than those in control group ( P0.05 ) .
Bone marrow proliferation of hUCBDSCs and hUCBDMSCs were better than those in control group , and hUCBDSCs were superior to hUCBDMSCs .
Conclusion : 1 . The hUCBDSCs and hUCBDMSCs can be used as a safe and effective method for repairing hematopoietic microenvironment damage after radiotherapy / chemotherapy . Conclusion : 1 . The hUCBDSCs and hUCBDMSCs can be used as a safe and effective method for repairing hematopoietic microenvironment injury after radiotherapy / chemotherapy . Conclusion : 1 . The hUCBDSCs and hUCBDMSCs can be used as a safe and effective method for repairing hematopoietic microenvironment injury after radiotherapy / chemotherapy .
The results showed that hUCBDSCs were superior to hUCBDMSCs .
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R733.72;R-332

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