本文選題：人臍血間充質(zhì)干細(xì)胞 + 創(chuàng)傷性腦損傷��； 參考：《華北煤炭醫(yī)學(xué)院》2009年碩士論文

【摘要】：目的:靜脈注射人臍血間充質(zhì)干細(xì)胞(Human umbilical cord blood mesenchymal stem cell,HUCBMSCs)治療大鼠創(chuàng)傷性腦損傷(Traumatic brain injury,TBI),觀察治療前后大鼠神經(jīng)損傷嚴(yán)重程度變化、神經(jīng)生長因子(Never growth factor,NGF)、腦源性神經(jīng)營養(yǎng)因子(Brain-derived neurotrophic factor,BDNF)表達(dá)以及凋亡細(xì)胞數(shù)量的改變,旨在說明HUCBMSCs通過影響損傷局部NGF、BDNF表達(dá),從而改善損傷局部微環(huán)境,減少細(xì)胞凋亡,促進(jìn)神經(jīng)功能恢復(fù)。 方法:取第4代HUCBMSCs,于注射前72小時,用5-溴-2-脫氧尿嘧啶核苷(5-bromo-2-deoxyuridine,Brdu)標(biāo)記,移植備用。標(biāo)記后的細(xì)胞用免疫細(xì)胞化學(xué)檢測細(xì)胞標(biāo)記率,用臺盼藍(lán)排斥試驗檢測標(biāo)記后細(xì)胞的活性。 取清潔級健康雄性SD大鼠90只隨機(jī)分為假傷組(30只)、損傷對照組(以下簡稱“對照組”)(30只)和注射人臍血間充質(zhì)干細(xì)胞治療組(以下簡稱“治療組”)(30只)。然后每組再隨機(jī)分為注射后3天、7天、14天、21天和28天組,每個亞組6只大鼠。采用改進(jìn)的Feeney自由落體方法制作大鼠TBI模型。 造模成功后24小時,治療組所有老鼠經(jīng)鼠尾靜脈注入3×106個HUCBMSCs,以1ml的PBS液溶解;假傷組及對照組所有老鼠經(jīng)鼠尾靜脈注入等體積的PBS液。3組大鼠均未用免疫抑制劑。于相應(yīng)時間點處死大鼠之前行神經(jīng)損傷嚴(yán)重程度評分(Neurological Severity Score Points,NSS)。取出標(biāo)本后,采用HE染色、免疫組化法、原位雜交法對TBI大鼠注射后不同時間點的腦組織在光鏡下行形態(tài)學(xué)變化、Brdu、BDNF和NGF因子、以及細(xì)胞凋亡的觀測。 結(jié)果:剛傳代的細(xì)胞呈圓形,很快貼壁,伸展為三角形、多角形,并逐漸變成梭形,傳代24h內(nèi)完全貼壁,貼壁細(xì)胞均勻分布,恢復(fù)原來形態(tài),3～5天迅速增殖,約6～7天長滿培養(yǎng)瓶底。傳至第4代細(xì)胞形態(tài)較先更均勻,排列更有序。取傳至第4代的HUCBMSCs摻入Brdu,72h后免疫組化染色顯示80～90%細(xì)胞Brdu表達(dá)陽性。表明HUCBMSCs的標(biāo)記率達(dá)到80～90%。移植后剩余細(xì)胞行臺盼藍(lán)染色,結(jié)果顯示90%以上細(xì)胞存活。 大鼠NSS評分顯示,自注射HUCBMSCs后第7天,各時間點治療組的神經(jīng)功能改善均顯著優(yōu)于對照組(P0.05)。 光鏡下假傷組大鼠腦組織細(xì)胞結(jié)構(gòu)基本正常;對照組大鼠受損皮層可見組織結(jié)構(gòu)破壞、脫落,神經(jīng)元變性,胞體皺縮。治療組光鏡下病理改變較損傷對照組輕微。 治療組可見Brdu標(biāo)記細(xì)胞在損傷周邊區(qū)明顯聚集并存活。隨著時間延長,Brdu標(biāo)記細(xì)胞逐漸減少。 假傷組僅有極少數(shù)神經(jīng)細(xì)胞呈TUNEL陽性。對照組腦損傷周邊區(qū)出現(xiàn)大量TUNEL染色陽性細(xì)胞,和假傷組比較有顯著差異(P0.05),相同時間點治療組腦損傷周邊區(qū)TUNEL陽性細(xì)胞明顯減少,但仍顯著高于假傷組(P0.05)。隨著時間推移,對照組和治療組TUNEL陽性細(xì)胞數(shù)量逐漸減少。 假傷組僅有少量的BDNF、NGF陽性表達(dá)細(xì)胞。對照組BDNF及NGF表達(dá)顯著增加,以損傷周邊區(qū)最為明顯,約注射后14天達(dá)到高峰,之后逐漸下降,但仍高于假傷組。治療組BDNF、NGF陽性細(xì)胞的表達(dá)明顯增加,與相同時間點的對照組比較有顯著差異(P0.05),注射后14天達(dá)高峰,21d、28d陽性表達(dá)明顯下降,但仍高于相同時間點的對照組。 結(jié)論: 1.經(jīng)尾靜脈注射HUCBMSCs可以遷移至腦創(chuàng)傷灶的周邊區(qū)。 2.未用免疫抑制劑下,HUCBMSCs在免疫功能健全的異種(大鼠)腦內(nèi)至少能存活4周,且大鼠未發(fā)生移植相關(guān)的死亡。 3.注入HUCBMSCs能促進(jìn)腦創(chuàng)傷灶周圍區(qū)神經(jīng)元功能恢復(fù),并且NSS評分下降,BDNF、NGF基因蛋白增多,BDNFmRNA表達(dá)增強(qiáng),神經(jīng)元凋亡減少。
[Abstract]:Objective: intravenous injection of human umbilical cord blood mesenchymal stem cells (Human umbilical cord blood mesenchymal stem cell, HUCBMSCs) in the treatment of traumatic brain injury in rats (Traumatic brain injury, TBI), the changes in the severity of nerve injury in the rats before and after treatment, the neurotrophic factor, and brain derived neurotrophic factor (brain-derived neurotrophic factor) The expression of rived neurotrophic factor, BDNF) and the changes in the number of apoptotic cells are designed to indicate that HUCBMSCs can improve the local microenvironment, decrease the apoptosis and promote the recovery of nerve function by affecting the local NGF and BDNF expression of damage.
Methods: fourth generations of HUCBMSCs, 72 hours before the injection, were labeled with 5- bromine -2- deoxy uridine (5-bromo-2-deoxyuridine, Brdu). The labeled cells were labeled with immunocytochemistry, and the activity of the labeled cells was detected by trypan blue rejection test.
90 healthy male SD rats were randomly divided into a false injury group (30 rats), the injury control group (the following "control group") (30) and the injection of human umbilical cord blood mesenchymal stem cells (the following "treatment group") (30). Then each group was randomly divided into 3 days after the injection, 7 days, 14 days, 21 days and 28 days, and 6 rats in each subgroup. The improved Feeney free fall method was used to produce rat TBI model.
24 hours after the success of the model, all rats in the treatment group were injected with 3 x 106 HUCBMSCs into the tail vein of the rat and dissolved in the PBS solution of 1ml. All rats in the false injury group and the control group were injected with the same volume of the.3 group of the PBS solution in the tail vein of the rat. The severity score of the nerve injury (Neurological Se) before the rats were executed at the corresponding time point. Verity Score Points, NSS). After taking out the specimens, using HE staining, immunohistochemistry and in situ hybridization, the morphological changes of the brain tissues at different time points after injection of TBI rats were observed under light microscopy, Brdu, BDNF and NGF, and the observation of cell apoptosis.
Results: the cells in the new generation were round, quickly adhered to the wall, stretched into triangular, polygonal, and gradually turned into spindle shape. The cells in 24h were completely adhered to the wall, the adherent cells were evenly distributed, restored to the original form, proliferated rapidly in 3~5 days, and about 6~7 days were filled with the bottom of the culture bottle. The form of the fourth generation of fine cells was more uniform and ordered more orderly. Then to the fourth generation of HUCB MSCs was added into Brdu, and the expression of Brdu in 80 ~ 90% cells was positive after 72h. It showed that the labeling rate of HUCBMSCs reached 80 ~ 90%. after transplantation of trypan blue, and the results showed that more than 90% cells survived.
The NSS score of rats showed that the neurological function improvement of the treatment group at each time point was significantly better than that of the control group on the seventh day after injection of HUCBMSCs (P0.05).
The structure of brain tissue in the rats with false injury under light microscope was basically normal, and the damaged cortex of the control group could see the destruction of tissue structure, the degeneration of the neurons, the degeneration of the neurons and the contraction of the cells. The pathological changes under the light microscope in the treatment group were slightly more than that of the control group.
In the treatment group, Brdu labeled cells obviously clustered and survived in the injured peripheral area, and the Brdu labeled cells decreased gradually as time went on.
Only a few nerve cells in the false injury group were TUNEL positive. There were a large number of TUNEL positive cells in the peripheral area of the control group, and there was a significant difference between the false group and the false group (P0.05). The TUNEL positive cells in the peripheral area of the brain injury at the same time point were significantly reduced, but still significantly higher than those in the false injury group (P0.05). The number of TUNEL positive cells in the group decreased gradually.
There were only a small amount of BDNF and NGF positive cells in the false injury group. The expression of BDNF and NGF in the control group increased significantly, which was the most obvious in the peripheral area, and reached the peak at 14 days after the injection, and then decreased gradually, but it was still higher than that in the false group. The expression of BDNF, NGF positive cells in the treatment group was significantly increased, and there was a significant difference compared with the control group at the same time point (P0.05 After injection, the positive expression of 21d and 28d decreased significantly in 14 Tianda peak, but still higher than that in the control group at the same time point.
Conclusion:
1. HUCBMSCs injected into caudal vein can migrate to the peripheral area of the brain injury foci.
2. without immunosuppressive agents, HUCBMSCs could survive at least 4 weeks in the brain of a rat with immunological function, and there was no death related to transplantation.
3. injection of HUCBMSCs could promote the recovery of neuron function around the brain wound area, and the NSS score decreased, BDNF, NGF gene protein increased, the expression of BDNFmRNA was enhanced, and the neuron apoptosis decreased.

【學(xué)位授予單位】：華北煤炭醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329.2

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