本文選題：臍帶間充質(zhì)干細(xì)胞 + 細(xì)胞移植　； 參考：《河北醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：建立大鼠液壓沖擊顱腦損傷模型,探討人臍帶間充質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells, hUC-MSCs)移植對(duì)顱腦損傷大鼠神經(jīng)功能恢復(fù)的影響以及移植細(xì)胞的存活、遷移和神經(jīng)分化情況，為其在顱腦損傷治療的應(yīng)用提供理論依據(jù)。 方法：取足月妊娠剖宮產(chǎn)健康新生兒的臍帶，利用酶消化法和貼壁法獲得原代細(xì)胞，傳3~5代備用。收集消化后的細(xì)胞，流式細(xì)胞學(xué)檢測(cè)CD45PE、CD90PE、 CD105PE、CD73PE、CD11b PE和CD34FITC、CD19FITC， HLA-DR PE，并以小鼠IgG1-PE和小鼠IgG1-FITC作為同型對(duì)照。 選取成年健康雄性Sprague-Dawley (SD)大鼠，體重約260g～320g，制作液壓沖擊顱腦損傷模型。模型制備成功24小時(shí)后，采取改良神經(jīng)功能評(píng)分（modified Neurological Severity Score, mNSS）對(duì)其進(jìn)行神經(jīng)功能評(píng)價(jià)（Table1），選取評(píng)分為7～12分（中度）的50只大鼠進(jìn)入實(shí)驗(yàn)。將其隨機(jī)分為三組：⑴hUC-MSCs移植組：20只，細(xì)胞移植前用噻唑藍(lán)[3-(4,5)-dimethylthiahiazol-2-y1-2,5-diphenytetrazolium bromide，MTT]檢測(cè)細(xì)胞活力。將第5代hUC-MSCs移植到大鼠顱腦損傷部位。⑵磷酸鹽緩沖液（phosphate buffered saline, PBS）組：20只，用PBS替代hUC-MSCs注射到顱腦損傷部位。⑶單純顱腦損傷：10只，行開骨窗和顱腦損傷處理，但不移植hUC-MSCs。另外，再隨機(jī)選取10只大鼠為⑷假手術(shù)組，只開骨窗，不行顱腦損傷處理，亦不行細(xì)胞移植。 四組大鼠分別采用改良的神經(jīng)功能評(píng)分(modified neurologicalseverity score，mNSS)在hUC-MSCs移植或PBS注射后的第１天、7天、14天、21天、28天進(jìn)行神經(jīng)功能評(píng)分，觀察其神經(jīng)功能改善情況。評(píng)分結(jié)束后采用RT-PCR技術(shù)檢測(cè)移植細(xì)胞膠質(zhì)纖維酸性蛋白(glial fibrillaryacidic protein,GFAP)、神經(jīng)元特異性烯醇化酶(neuron specific enolase,NSE)、巢蛋白(Nestin)以及髓鞘堿性蛋白(myelin basic protein，MBP)的表達(dá)情況，并進(jìn)行比較。 結(jié)果： 細(xì)胞接種4～6h后觀察到少量細(xì)胞貼壁，，12h以后大部分細(xì)胞貼壁。接種7d時(shí)，細(xì)胞匯合達(dá)到80％～90％，消化后按1:2或1:3的比例傳代。細(xì)胞傳代后，形態(tài)均一，生長(zhǎng)迅速，3-4d可傳一代。流式細(xì)胞學(xué)檢測(cè)結(jié)果顯示：hUC-MSCs表達(dá)間充質(zhì)干細(xì)胞表面抗原CD105、CD73、CD90，不表達(dá)B型淋巴細(xì)胞表面抗原CD19、造血前體細(xì)胞表面抗原CD34、白細(xì)胞表面抗原CD45、中性粒細(xì)胞表面抗原CD11b和HLA-DR，表示該細(xì)胞是hUC-MSCs且具備異體移植的可行性。細(xì)胞移植前采用MTT法檢測(cè)hUC-MSCs細(xì)胞活力好，臺(tái)盼藍(lán)染色顯示細(xì)胞存活率達(dá)95%以上。 hUC-MSCs移植后28d內(nèi)，hUC-MSCs移植組死亡2只，死亡率為10.0%；PBS組死亡6只，死亡率為30.0%；單純顱腦損傷組死亡4只，死亡率40.0%。hUC-MSCs移植組死亡率低于單純顱腦損傷組和PBS組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 神經(jīng)功能評(píng)分結(jié)果（Table3）顯示，hUC-MSCs移植組隨著時(shí)間的變化神經(jīng)功能恢復(fù)優(yōu)于單純顱腦損傷組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 hUC-MSCs移植28天，神經(jīng)功能評(píng)分結(jié)束后，取新鮮腦組織提取總RNA,應(yīng)用RT-PCR從分子生物學(xué)證實(shí)了移植的hUC-MSCs在大鼠顱腦損傷部位存活并表達(dá)GFAP、NSE、Nestin，未表達(dá)MBP。 結(jié)論： 1人臍帶間充質(zhì)干細(xì)胞移植促進(jìn)大鼠顱腦損傷后神經(jīng)功能的恢復(fù)。 2移植的hUC-MSCs在大鼠顱腦損傷部位存活并表達(dá)GFAP、NSE、Nestin，未表達(dá)MBP。
[Abstract]:Objective: to establish a model of hydraulic impact craniocerebral injury in rats and to explore the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) transplantation on the recovery of neural function in rats with craniocerebral injury and the survival, migration and differentiation of the transplanted cells in order to provide a theory for the application of the brain injury in the treatment of brain injury. Basis.
Methods: the umbilical cord of the healthy newborn in the term of cesarean section was taken. The original cells were obtained by enzyme digestion and adherence method. The cells were collected for 3~5 generation. The cells after digestion were collected, and the flow cytometry was used to detect CD45PE, CD90PE, CD105PE, CD73PE, CD11b PE and CD34FITC, CD19FITC, HLA-DR PE, and the mice IgG1-PE and mice were used as the same control.
The adult healthy male Sprague-Dawley (SD) rats were selected and weighed about 260g to 320G to make a model of hydraulic shock craniocerebral injury. After 24 hours of successful model preparation, the modified neural function score (modified Neurological Severity Score, mNSS) was used to evaluate the nerve function of the brain injury (Table1), and 50 rats with a score of 7~12 (moderate) were selected. The experiment was randomly divided into three groups: (1) hUC-MSCs transplantation group: 20, cell viability was detected by thiazolium [3- (4,5) -dimethylthiahiazol-2-y1-2,5-diphenytetrazolium bromide and MTT] before transplantation. Fifth generation hUC-MSCs was transplanted into the brain injury site of rats. (2) phosphate buffer solution (phosphate buffered saline, PBS) group: 20 PBS instead of hUC-MSCs was injected into the site of craniocerebral injury. (3) simple craniocerebral injury: 10 rats were treated with open bone window and craniocerebral injury, but no transplantation of hUC-MSCs., 10 rats were randomly selected as a sham operation group, only open bone window, no craniocerebral injury treatment, and no cell transplantation.
The four groups of rats were treated with improved neural function score (modified neurologicalseverity score, mNSS) on the first day after hUC-MSCs transplantation or PBS injection, 7 days, 14 days, 21 days and 28 days to evaluate the neural function improvement. After the score, the RT-PCR technique was used to detect the glial fibrillary acidic protein (glia) of the transplanted cells (glia). L fibrillaryacidic protein, GFAP), the expression of neuron specific enolase (neuron specific enolase, NSE), nestin (Nestin) and myelin basic protein (myelin basic protein), and compared.
Result錛

本文編號(hào)：2022156