本文選題：人臍血源基質(zhì)細(xì)胞 + 人臍血源間充質(zhì)干細(xì)胞�。� 參考：《瀘州醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的：本課題采用密度梯度離心收集獲得人臍血單個核細(xì)胞（mononuclear cells, MNCs），經(jīng)過傳代培養(yǎng)得到人臍血源基質(zhì)細(xì)胞（humanumbilical cord blood-derived stromal cells, hUCBDSCs）、人臍血源間充質(zhì)干細(xì)胞（human umbilical cord blood-derived mesenchymal stem cells,hUCBDMSCs）；觀察兩類細(xì)胞形態(tài)結(jié)構(gòu)、生長增殖特點(diǎn)，流式細(xì)胞儀技術(shù)測得兩類細(xì)胞的表面抗原；經(jīng)DiI標(biāo)記后分別輸注入造血功能損傷模型小鼠模型體內(nèi)，動態(tài)檢測小鼠靜脈血紅細(xì)胞、白細(xì)胞、血小板、血紅蛋白濃度，了解外周血血象恢復(fù)情況；動態(tài)檢測兩類細(xì)胞對骨髓造血微環(huán)境的修復(fù)情況，深入比較人臍血源基質(zhì)細(xì)胞與其間充質(zhì)干細(xì)胞恢復(fù)造血的能力，達(dá)到確立在臨床可使用人臍血源基質(zhì)細(xì)胞即可恢復(fù)造血這一理論體系提供了動物實(shí)驗(yàn)依據(jù)，解決了臨床上因使用促造血生長因子帶來的副作用及產(chǎn)生昂貴費(fèi)用這一實(shí)際問題；也給長期探索早期造血恢復(fù)方法的血液學(xué)工作者帶來了新的思路。方法：1.細(xì)胞分離與培養(yǎng)：采用密度梯度離心收集獲得臍血單個核細(xì)胞，用DMEM/F-12、MSCM培養(yǎng)體系分別進(jìn)行單個核細(xì)胞的體外培養(yǎng)，得到純度較高的hUCBDSCs及hUCBDMSCs，倒置顯微鏡下動態(tài)觀察兩類細(xì)胞生長增殖情況及形態(tài)特點(diǎn)，利用流式細(xì)胞技術(shù)檢測兩類細(xì)胞表面抗原；2.DiI標(biāo)記兩類細(xì)胞進(jìn)行示蹤；3.建立小鼠造血損傷模型：X射線輻照BALB/c小鼠（總劑量5Gy，吸收率100cGy/min，時間5min，距離100cm）；4.實(shí)驗(yàn)分組：將造型損傷模型小鼠隨機(jī)分為3組：hUCBDSCs組、hUCBDMSCs組、對照組；5.分別經(jīng)小鼠尾靜脈移植注入hUCBDSCs（1.0×106/只）、hUCBDMSCs（1.0×106/只）、生理鹽水（0.2ml），動態(tài)記錄三組小鼠的精神狀態(tài)、飲食情況等一般生理狀態(tài)及生存狀況，計(jì)算各組生存率；分別于3d、7d、14d取靜脈血檢測紅細(xì)胞、白細(xì)胞、血小板、血紅蛋白，了解外周血血象恢復(fù)情況；3d、7d、14d分別進(jìn)行骨髓涂片觀察兩類細(xì)胞在小鼠骨髓歸巢情況、骨髓活檢了解兩類細(xì)胞對骨髓造血微環(huán)境損傷的修復(fù)作用；6.統(tǒng)計(jì)學(xué)分析：應(yīng)用SPSS22.0統(tǒng)計(jì)分析軟件對所獲得的數(shù)據(jù)進(jìn)行處理，計(jì)量數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(ˉx±s)表示，各組間采用單因素方差分析，兩組間數(shù)據(jù)比較應(yīng)用t檢驗(yàn)，檢驗(yàn)水準(zhǔn)α設(shè)定為0.05，P0.05差異有統(tǒng)計(jì)學(xué)意義；計(jì)數(shù)資料采用率描述。結(jié)果：1.采用密度梯度離心可獲得單個核細(xì)胞，經(jīng)體外傳代培養(yǎng)得到純度較高的hUCBDMSCs及hUCBDSCs；MSCM培養(yǎng)體系培養(yǎng)原代細(xì)胞，24h后細(xì)胞呈不規(guī)則形、類圓形，分布均勻，胞體小，透明；48h后細(xì)胞貼壁，數(shù)量增多，部分細(xì)胞開始向兩極伸展；7-12d細(xì)胞以梭形為主，集落樣分布，細(xì)胞內(nèi)顆粒少，透明，細(xì)胞間界限清楚；14-16d可鋪滿瓶底；傳代后細(xì)胞形態(tài)一致，生長增殖較原代快，經(jīng)1-2d潛伏期后進(jìn)入對數(shù)生長期，呈均勻細(xì)長梭形，6-7d后進(jìn)入平臺期。F-12培養(yǎng)體系培養(yǎng)原代細(xì)胞，24h后細(xì)胞呈類圓形、不規(guī)則形，分布均勻，胞質(zhì)透明；48h后細(xì)胞數(shù)量增多，貼壁生長；7-12d細(xì)胞集落樣分布，形態(tài)多樣，以類圓形、扁形為主，細(xì)胞間界限清楚，胞質(zhì)透明，顆粒少；傳代后細(xì)胞生長較原代快，經(jīng)1-2d潛伏期后進(jìn)入對數(shù)生長期，呈圓形或扁形，6d后進(jìn)入平臺期。兩類細(xì)胞生長曲線變化趨勢一致：傳代后1-2d細(xì)胞處于潛伏期，生長增殖緩慢；48h后細(xì)胞指數(shù)增長，增殖、活力旺盛，進(jìn)入對數(shù)生長期；6-7d后細(xì)胞生長增殖明顯減緩，由對數(shù)生長期進(jìn)入平臺期。hUCBDSCs對數(shù)生長期前期增殖速度較hUCBDMSCs緩慢，后期增長速度快于hUCBDMSCs。2.流式細(xì)胞術(shù)檢測細(xì)胞表面抗原的表達(dá)，hUCBDMSCs表達(dá)CD29、CD44、CD106、STRO-1，不表達(dá)CD34、CD45；hUCBDSCs表達(dá)CD44、CD45、Lm、Fn，不表達(dá)CD29、STRO-1，兩類細(xì)胞表面抗原的表達(dá)與大量研究結(jié)果一致，證實(shí)DMEM/F-12培養(yǎng)體系可培養(yǎng)出hUCBDSCs，MSCM培養(yǎng)體系可培養(yǎng)出hUCBDMSCs。3.模型的建立：非致死劑量輻照BALB/c小鼠（劑量5Gy，能量6MeV，距離100cm，，吸收率100cGy/min，時間5min），可造成小鼠急性造血功能損傷。4.DiI標(biāo)記第三代hUCBDMSCs、hUCBDSCs，兩類細(xì)胞在激光共聚焦顯微鏡下顯橙紅色熒光；形態(tài)與普通倒置相差顯微鏡一致；兩類細(xì)胞標(biāo)記率均為100%。5.血象的恢復(fù)情況：動態(tài)監(jiān)測血常規(guī)，結(jié)果顯示移植后3d，三組小鼠白細(xì)胞、血小板、紅細(xì)胞計(jì)數(shù)及血紅蛋白濃度均無顯著差異(P0.05)；移植后第7d，hUCBDSCs組及hUCBDMSCs組白細(xì)胞、血小板、紅細(xì)胞、血紅蛋白恢復(fù)情況均顯著高于對照組(P0.05)，且hUCBDSCs組白細(xì)胞、血小板、紅細(xì)胞均顯著高于hUCBDMSCs組(P0.05)，血紅蛋白無濃度明顯差異(P0.05)。移植后14d，hUCBDSCs組及hUCBDMSCs組白細(xì)胞、血小板、紅細(xì)胞、血紅蛋白恢復(fù)均顯著高于對照組(P0.05)，且hUCBDSCs組白細(xì)胞、血小板計(jì)數(shù)及血紅蛋白濃度顯著高于hUCBDMSCs組(P0.05)，紅細(xì)胞計(jì)數(shù)無明顯差異(P0.05)。6.兩類細(xì)胞的歸巢：動態(tài)監(jiān)測骨髓示蹤細(xì)胞，結(jié)果顯示兩類細(xì)胞均可在移植后期穩(wěn)定定植于骨髓，且hUCBDSCs組較hUCBDMSCs組先歸巢于骨髓(P0.05)。7.骨髓病理切片：移植后3d三組小鼠骨髓均出現(xiàn)造血功能抑制，有核細(xì)胞增生不良，非造血細(xì)胞比例增加；移植后7d三組小鼠造血功較前均有恢復(fù)，有核細(xì)胞成簇分布，hUCBDSCs組、hUCBDMSCs組骨髓增生均優(yōu)于對照組，且hUCBDSCs組優(yōu)于hUCBDMSCs組；移植后14d對照組骨髓造血功能恢復(fù)情況較前無明顯改變，hUCBDSCs組與hUCBDMSCs組造血功能明顯恢復(fù)，有核細(xì)胞增生活躍，人臍血源基質(zhì)細(xì)胞組可見大量巨核細(xì)胞。提示臍血干細(xì)胞移植可作為修復(fù)放/化療后造血微環(huán)境損傷的一種安全有效方法，具有潛在的臨床應(yīng)用前景。結(jié)論：1.利用密度梯度離心，體外培養(yǎng)可獲得高純度的hUCBDMSCs及hUCBDSCs。2.hUCBDSCs、hUCBDMSCs移植入小鼠體內(nèi)后，可明顯促進(jìn)白細(xì)胞、血小板、紅細(xì)胞恢復(fù)，且hUCBDSCs恢復(fù)血象作用更強(qiáng)，骨髓抑制程度更輕。3.hUCBDSCs、hUCBDMSCs移植入造血損傷模型小鼠體內(nèi)后，均可穩(wěn)定定植于骨髓，且hUCBDSCs較hUCBDMSCs先歸巢于骨髓；兩類細(xì)胞對骨髓造血功能恢復(fù)均有明顯促進(jìn)作用，且hUCBDSCs優(yōu)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 鹵 standard deviation ( 藟x 鹵 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 .
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R733.72;R-332

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 林京玉;應(yīng)翔宇;鐘玉緒;丁日高;榮曙;袁本利;余壽忠;;芥子氣致小鼠耳廓皮膚損傷模型的建立[J];軍事醫(yī)學(xué)科學(xué)院院刊;2006年01期

2 苗明三;動物剪皮損傷模型制造方法[J];河南中醫(yī);1997年03期

3 顏培華,李鳳芝,劉嘉瀛,楊增仁;血管內(nèi)皮細(xì)胞冷損傷模型的建立[J];解放軍預(yù)防醫(yī)學(xué)雜志;2005年02期

4 蘇楠;陳林;;小鼠骨損傷模型在骨修復(fù)機(jī)制研究中的應(yīng)用[J];國際病理科學(xué)與臨床雜志;2007年03期

5 李偉萍,朱志祥,劉天一,許曉光,黃鐵軍,何廣仁;兔高壓電非熱性損傷模型的建立[J];中華燒傷雜志;2000年06期

6 程化坤;王麗虹;李波;劉恩重;;體外培養(yǎng)大鼠腦皮質(zhì)神經(jīng)元離心損傷模型的建立[J];中華神經(jīng)外科雜志;2005年12期

7 張旭,梅曉云 ,卞慧敏 ,繆愛琴,張超英;骨骼肌細(xì)胞損傷模型的建立[J];解放軍藥學(xué)學(xué)報(bào);2003年02期

8 朱波;中樞神經(jīng)系統(tǒng)缺血缺氧損傷模型[J];國外醫(yī)學(xué).麻醉學(xué)與復(fù)蘇分冊;2002年01期

9 盧奕,吳智遠(yuǎn),惠國楨,郭禮和,暨荀鶴;一種癥狀性大鼠腦損傷模型的建立[J];蘇州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年05期

10 熊華花;鄧志云;李學(xué)東;;周圍神經(jīng)電損傷模型的制備[J];江西醫(yī)學(xué)院學(xué)報(bào);2006年02期

相關(guān)會議論文 前10條

1 闕海萍;劉少君;;大鼠脊髓原代神經(jīng)元培養(yǎng)和損傷模型的建立[A];中國生理學(xué)會第21屆全國代表大會暨學(xué)術(shù)會議論文摘要匯編[C];2002年

2 唐占英;周泉;李晨光;王擁軍;周重建;施杞;;大鼠頸神經(jīng)背根節(jié)壓迫性損傷模型的設(shè)計(jì)與建立[A];中華中醫(yī)藥學(xué)會骨傷分會第四屆第二次會議論文匯編[C];2007年

3 胡英國;盧文波;陳明;嚴(yán)鵬;周創(chuàng)兵;;巖石爆破損傷模型的比選與改進(jìn)[A];第3屆全國工程安全與防護(hù)學(xué)術(shù)會議論文集[C];2012年

4 郭麗麗;王階;陳雯;周婷婷;;離體心臟缺血再灌時間與損傷模型的合理評價[A];2011年中華中醫(yī)藥學(xué)會心病分會學(xué)術(shù)年會暨北京中醫(yī)藥學(xué)會心血管病專業(yè)委員會年會論文集[C];2011年

5 沈憶文;趙子琴;陳憶九;閻祖康;顧云菊;吳文慶;廖燕萍;;體外培養(yǎng)人成纖維細(xì)胞(FBs)損傷模型的建立及其細(xì)胞免疫組織化學(xué)研究[A];全國第六次法醫(yī)學(xué)術(shù)交流會論文摘要集[C];2000年

6 張曦;陳幸華;司英健;劉耀;高蕾;高力;彭賢貴;王慶余;;裸鼠造血微環(huán)境損傷模型的建立[A];第11次中國實(shí)驗(yàn)血液學(xué)會議論文匯編[C];2007年

7 陳琢;焦新福;葉望云;舒滬英;;血瘀證大鼠紅細(xì)胞損傷模型的研制與運(yùn)用[A];第五次全國中西醫(yī)結(jié)合血瘀證及活血化瘀研究學(xué)術(shù)大會論文匯編[C];2001年

8 鄭黎燕;王國華;闕海萍;郁毅剛;馬振蓮;李欣;劉少君;;Nogo及其NgR受體在神經(jīng)細(xì)胞及損傷模型中的表達(dá)[A];中國生理學(xué)會第21屆全國代表大會暨學(xué)術(shù)會議論文摘要匯編[C];2002年

9 唐占英;周泉;李晨光;王擁軍;周重建;施杞;;大鼠頸神經(jīng)背根節(jié)壓迫性損傷模型的設(shè)計(jì)與建立[A];第十六屆全國中西醫(yī)結(jié)合骨傷科學(xué)術(shù)研討會暨中西醫(yī)結(jié)合手法治療骨傷科疾病新進(jìn)展學(xué)習(xí)班論文匯編[C];2008年

10 劉銳;鞠躬;;還原論式大鼠脊髓急性全橫斷損傷模型的建立[A];中國神經(jīng)科學(xué)學(xué)會第六屆學(xué)術(shù)會議暨學(xué)會成立十周年慶祝大會論文摘要匯編[C];2005年

相關(guān)碩士學(xué)位論文 前6條

1 胡蕾;人臍血源基質(zhì)細(xì)胞對造血損傷模型小鼠體內(nèi)造血調(diào)控作用的研究[D];瀘州醫(yī)學(xué)院;2014年

2 祁玉婷;急性高原缺氧損傷模型的建立及腦線粒體差異蛋白表達(dá)研究[D];河北醫(yī)科大學(xué);2011年

3 張沛;基于GTN損傷模型的鋼節(jié)點(diǎn)斷裂預(yù)測研究[D];北京交通大學(xué);2014年

4 劉文濤;考慮混合強(qiáng)化準(zhǔn)則的GTN損傷模型[D];北京交通大學(xué);2014年

5 侯丕吉;型鋼高強(qiáng)高性能混凝土框架柱地震損傷模型研究[D];西安建筑科技大學(xué);2009年

6 史麗軍;考慮損傷恢復(fù)的鹽巖流變損傷模型[D];河北工業(yè)大學(xué);2014年

本文編號：2065711