本文選題：帕金森氏癥 + 人源神經(jīng)干細(xì)胞�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2014年博士論文

【摘要】：帕金森氏癥是常見的中樞神經(jīng)退行性疾病,在發(fā)達(dá)國家60歲以上人口中患病率約2%,在我國主要城市老齡人口中患病率相仿。帕金森氏癥主要臨床表現(xiàn)包括靜止性振顫、僵硬、動(dòng)作遲緩、姿勢(shì)反射異常等,嚴(yán)重者喪失生活自理能力。相對(duì)其他神經(jīng)退行性疾病,帕金森氏癥有一整套包括藥物和深部電極刺激手術(shù)在內(nèi)的治療方案,短期內(nèi)可改善癥狀,但均不能逆轉(zhuǎn)神經(jīng)病理改變,而長期用藥帶來的異動(dòng)癥等副作用顯著。上世紀(jì)八十年代起,干細(xì)胞與再生醫(yī)學(xué)的發(fā)展給該病的治療帶了新的思路,科學(xué)家利用人胚胎干細(xì)胞和基因工程技術(shù),將能夠表達(dá)多巴胺的胚胎干細(xì)胞移植到神經(jīng)退變的中腦黑質(zhì)或者丘腦紋狀體區(qū),能夠長期改善癥狀,并已用于臨床實(shí)驗(yàn)。但由于胚胎干細(xì)胞的來源和倫理學(xué)限制,未能廣泛開展,因而研究的熱點(diǎn)轉(zhuǎn)向間充質(zhì)干細(xì)胞、神經(jīng)干細(xì)胞等。在此,我們將體外培養(yǎng)的人類流產(chǎn)胚胎來源神經(jīng)干細(xì)胞系,移植入MPTP誘導(dǎo)的帕金森病小鼠模型紋狀體區(qū)域,以了解小鼠疾病微環(huán)境中此人源神經(jīng)干細(xì)胞系的存活、生長狀況,并研究神經(jīng)干細(xì)胞移植對(duì)帕金森病小鼠運(yùn)動(dòng)功能的影響,并由此建立了一整套應(yīng)用人源神經(jīng)干細(xì)胞系治療小鼠神經(jīng)退行性疾病的研究體系。該研究在移植采用的細(xì)胞類型,移植方案的精確度,降低移植副損傷方面相對(duì)該領(lǐng)域同期研究有較好的表現(xiàn)。 目的：研究人源神經(jīng)干細(xì)胞移植入帕金森氏癥小鼠紋狀體后的生存狀況；研究疾病微環(huán)境中移植神經(jīng)干細(xì)胞的分化、遷移、凋亡等特性；研究移植入神經(jīng)干細(xì)胞與宿主神經(jīng)網(wǎng)絡(luò)是否形成聯(lián)系；研究移植入的神經(jīng)干細(xì)胞,是否能對(duì)帕金森病小鼠的運(yùn)動(dòng)癥狀起到改善作用；評(píng)估帕金森病小鼠不同運(yùn)動(dòng)障礙對(duì)人源神經(jīng)干細(xì)胞治療的敏感性及評(píng)估方法；為后期進(jìn)行不同動(dòng)物模型及轉(zhuǎn)基因干細(xì)胞的移植實(shí)驗(yàn)建立可行方案和流程。 研究內(nèi)容與技術(shù)路線： 1、建立穩(wěn)定的帕金森氏癥小鼠模型。采用經(jīng)典的MPTP帕金森病C57/B16小鼠模型,實(shí)驗(yàn)組按照每次MPTP20mg/kg體重(溶于0.9%生理鹽水)劑量,共進(jìn)行腹腔注射4次,每次間隔3小時(shí)。對(duì)照組腹腔注射相同劑量0.9%生理鹽水。 2、行為學(xué)評(píng)估。采用轉(zhuǎn)棒實(shí)驗(yàn)(Rota-Rod)和爬竿實(shí)驗(yàn)(Rod Climbing),分別建立帕金森病小鼠的運(yùn)動(dòng)障礙評(píng)估方法。 3、人源神經(jīng)干細(xì)胞培養(yǎng),用慢病毒將GFP導(dǎo)入細(xì)胞,使細(xì)胞帶有綠色熒光標(biāo)記。 4、神經(jīng)于細(xì)胞的立體定向注射。實(shí)驗(yàn)分組： 5、評(píng)估注入神經(jīng)干細(xì)胞在PD小鼠紋狀體微環(huán)境內(nèi)的生存、分化、遷移、凋亡、與宿主神經(jīng)網(wǎng)絡(luò)整合等現(xiàn)象。 6、用轉(zhuǎn)棒實(shí)驗(yàn)和爬竿實(shí)驗(yàn)對(duì)比帕金森病小鼠和對(duì)照組在治療前后、不同時(shí)期的運(yùn)動(dòng)癥狀變化情況,評(píng)估立體定向注射本身和干細(xì)胞移植對(duì)PD小鼠運(yùn)動(dòng)癥狀的影響。 結(jié)果： 1、小鼠以20mg/kg劑量,間隔3小時(shí),分四次注射MPTP后,小鼠3日內(nèi)死亡率20%,此后未再有死亡。一周后轉(zhuǎn)棒實(shí)驗(yàn)持續(xù)時(shí)間減少74.6%,癥狀能夠在2月內(nèi)保持穩(wěn)定。 2、干細(xì)胞立體定向注入實(shí)驗(yàn)組及對(duì)照組小鼠紋狀體,術(shù)后2日內(nèi)急性死亡率小于4%,2日后無死亡,一周后傷口愈合好,無感染。 3、觀察陰性對(duì)照1組(不做立體定向注射)、陰性對(duì)照2組(立體定向注射PBS緩沖液),兩組在注射后第1,2,3,4,5周時(shí)轉(zhuǎn)棒實(shí)驗(yàn)運(yùn)動(dòng)癥狀無顯著性差異,單純手術(shù)對(duì)PD小鼠運(yùn)動(dòng)癥狀無明顯影響。 4、對(duì)比實(shí)驗(yàn)組(立體定向注射神經(jīng)干細(xì)胞)、陰性對(duì)照2組(立體定向注射PBS緩沖液),術(shù)后前三周實(shí)驗(yàn)組轉(zhuǎn)棒持續(xù)時(shí)間明顯改善平均轉(zhuǎn)棒持續(xù)時(shí)間60.5s,82.5s,98.5s,分別較對(duì)照2組提高49.3%,120.1%,94.9%,此后實(shí)驗(yàn)組運(yùn)動(dòng)能力逐漸下降,顯示治療效果減退,至術(shù)后第5周與對(duì)照組已無顯著性差異。 5、細(xì)胞學(xué)實(shí)驗(yàn)顯示,植入人類成體神經(jīng)干細(xì)胞在小鼠紋狀體內(nèi)可以存活,并產(chǎn)生一定遷移,在注入2周后發(fā)現(xiàn)最遠(yuǎn)遷移距離900μm。Double Cortin染色顯示部分神經(jīng)干細(xì)胞已分化為新生神經(jīng)元,2周時(shí)比例約為40%,周圍大量神經(jīng)膠質(zhì)增生。注入2周時(shí)部分神經(jīng)干細(xì)胞已分化出軸突,Synaptophysin染色顯示部分軸突上已有突觸后膜結(jié)構(gòu),顯示移植干細(xì)胞已與宿主形成簡單神經(jīng)網(wǎng)絡(luò)。 6、至干細(xì)胞注射后第四周,移植細(xì)胞存活率已明顯降低,約為第一周時(shí)存活細(xì)胞的2%,與運(yùn)動(dòng)癥狀治療效果消退時(shí)間平行。 7、注入神經(jīng)干細(xì)胞Ki-67染色,顯示已分化的神經(jīng)元不表達(dá)Ki-67,不具備分裂潛能,致瘤可能性低。 結(jié)論： 1、MPTP藥物可誘導(dǎo)產(chǎn)生短期內(nèi)癥狀穩(wěn)定的帕金森氏癥C57/B16小鼠模型。該模型運(yùn)動(dòng)癥狀可以用轉(zhuǎn)棒實(shí)驗(yàn)及爬桿實(shí)驗(yàn)進(jìn)行評(píng)估。 2、人源神經(jīng)干細(xì)胞注入MPTP帕金森小鼠紋狀體區(qū)域,可以在4周以內(nèi)部分改善疾病小鼠的運(yùn)動(dòng)障礙,表現(xiàn)為轉(zhuǎn)棒實(shí)驗(yàn)持續(xù)時(shí)間延長,但無法長期保持。 3、人源神經(jīng)干細(xì)胞注入MPTP帕金森小鼠紋狀體區(qū)域后,能夠存活至4周左右。注入2周后已能分化為新生神經(jīng)元,并在紋狀體內(nèi)具備一定的遷移能力。 4、注入的神經(jīng)干細(xì)胞部分可分化為多巴胺能神經(jīng)元前體細(xì)胞,提示而未能分化為GABA能、谷氨酸能神經(jīng)元前體細(xì)胞,可能與紋狀體微環(huán)境相關(guān)。 5、神經(jīng)干細(xì)胞注入小鼠紋狀體后,能夠與宿主細(xì)胞之間形成突觸結(jié)構(gòu),顯示初步具備形成新生神經(jīng)網(wǎng)絡(luò)的基礎(chǔ)。
[Abstract]:Parkinson's disease is a common degenerative disease of the central nervous system. The prevalence rate is about 2% in the population over 60 years of age in the developed countries. The prevalence rate is similar in the aged population in the main cities of our country. The main clinical manifestations of Parkinson's disease include static tremor, stiffness, slow motion and abnormal postural reflex, and the serious people lose their self-care ability. Relative to them His neurodegenerative disease, Parkinson's disease has a complete set of treatments including drugs and deep electrode stimulation, which can improve symptoms in the short term but can not reverse the neuropathological changes, and the side effects of long-term drug use are significant. Since 80s, the development of stem cells and regenerative medicine gave the disease to the disease. With new ideas, scientists have used human embryonic stem cells and genetic engineering techniques to transplant dopamine derived embryonic stem cells into the mesencephalic substantia nigra or the thalamus striatum, which can improve symptoms for a long time and have been used in clinical trials. But because of the origin and ethical limitations of embryonic embryonic stem cells, they have not been widely used. As a result, the focus of the study turned to mesenchymal stem cells, neural stem cells, etc. here, we transplanted the cultured human aborted embryonic stem cell lines into the striatum area of the MPTP induced Parkinson's disease mouse model to understand the survival and growth of this human neural stem cell line in the microenvironment of the mice. The effect of neural stem cell transplantation on the motor function of Parkinson's disease mice was investigated, and a whole set of research system was established to treat the neurodegenerative diseases of mice by using human neural stem cell lines. Good performance.
Objective: To study the survival status of human neural stem cells transplanted into the striatum of Parkinson's disease mice; to study the differentiation, migration and apoptosis of the transplanted neural stem cells in the microenvironment of the disease; to investigate whether the transplanted neural stem cells are associated with the host neural network; to investigate whether the transplanted neural stem cells can be used for PAS. The exercise symptoms of Higginson's disease mice were improved, and the sensitivity and evaluation methods of different motor disorders in Parkinson's disease on human neural stem cells were evaluated, and a feasible scheme and process were established for different animal models and transgenic stem cells in the later period.
Research content and technical route:
1, a stable Parkinson's disease model was established. The classic MPTP Parkinson's disease C57/B16 mouse model was used. The experimental group was injected 4 times in the abdominal cavity for 3 hours each time according to the dose of MPTP20mg/kg body weight (dissolved in 0.9% saline). The control group was injected with the same dose of 0.9% physiological saline.
2, behavioral assessment. Using Rota-Rod and Rod Climbing, Parkinson's disease mice were established to evaluate their movement disorders.
3, human neural stem cells were cultured with lentivirus to transfuse GFP into cells, so that the cells were labeled with green fluorescence.
4, stereotactic injection of neurons.
5, we assessed the survival, differentiation, migration, apoptosis and host neural network integration of neural stem cells injected into the microstripe of PD mice.
6, the changes of motor symptoms in different periods before and after treatment were compared between Parkinson's disease mice and the control group, and the effects of stereotactic injection and stem cell transplantation on the movement symptoms of PD mice were evaluated.
Result錛，

本文編號(hào)：1906556