本文選題：缺氧缺血性腦損傷 + 間充質(zhì)干細(xì)胞　； 參考：《山東大學(xué)》2014年博士論文

【摘要】：研究背景 缺氧缺血性腦損傷(hypoxic-ischemic brain damage,HIBD)是由于圍產(chǎn)期窒息、缺氧、缺血引致的新生兒腦損傷性疾病,圍產(chǎn)期的窒息會(huì)導(dǎo)致3～5/1000的活產(chǎn)嬰兒患中度或重度缺氧缺血性腦病,常常導(dǎo)致新生兒死亡和遺留一些神經(jīng)功能障礙,如腦性癱瘓、癲癇以及智力低下。其中,腦癱是兒童期最主要的運(yùn)動(dòng)機(jī)能傷殘性疾病,可造成患兒終生的殘疾,HIBD的高發(fā)病率、致殘率給患兒本人、家庭和社會(huì)都造成了巨大的精神、經(jīng)濟(jì)負(fù)擔(dān)。 HIBD的發(fā)病過(guò)程是一個(gè)十分復(fù)雜的病理過(guò)程,是多種機(jī)制綜合參與的一系列連鎖反應(yīng)的結(jié)果,而目前確切的發(fā)病機(jī)制并不明確。目前新生兒HIBD的治療多采用高壓氧治療、神經(jīng)細(xì)胞營(yíng)養(yǎng)藥物、物理康復(fù)等措施,這對(duì)損傷程度相對(duì)較輕的患兒具有一定的效果,但對(duì)于中-重度腦病所造成的中樞神經(jīng)功能障礙難以奏效。因此尋求一種有效的治療手段對(duì)減輕HIBD患兒的病死率、致殘率具有非常重要的臨床意義。 近年的研究表明,間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)治療在HIBD的動(dòng)物試驗(yàn)及臨床前期試驗(yàn)中都取得了很大的進(jìn)展,目前研究較多的是骨髓MSCs,但是BMSCs存在干細(xì)胞含量少、增殖能力和分化潛能減弱、有創(chuàng)操作、病毒感染的機(jī)率增高等局限性,使其臨床應(yīng)用和推廣受到了限制。本研究證實(shí)從孕鼠胎盤(pán)可以分離、培養(yǎng)胎盤(pán)來(lái)源MSCs (placenta-derived mesenchymal stem cells, PD-MSCs),經(jīng)鑒定符合間充質(zhì)干細(xì)胞鑒定標(biāo)準(zhǔn),驗(yàn)證了從大鼠胎盤(pán)獲得MSCs是完全可行的,而PD-MSCs因其自身的優(yōu)點(diǎn),將來(lái)可能會(huì)成為一種理想的種子細(xì)胞,具有極大的應(yīng)用潛能。 HIBD的發(fā)病機(jī)制涉及炎癥反應(yīng)、氧化應(yīng)激損傷、腦組織能量代謝紊亂等多個(gè)環(huán)節(jié)。缺血后的炎癥反應(yīng)已成為研究的焦點(diǎn),炎性細(xì)胞可以滲入到腦實(shí)質(zhì)并釋放多種神經(jīng)毒性物質(zhì)造成炎癥級(jí)聯(lián)反應(yīng),多種細(xì)胞因子參與其中,輔助T細(xì)胞17(T helper cell17, Th17)和調(diào)節(jié)性T細(xì)胞(regulatory T cells,Treg)也在HIBD的發(fā)生、發(fā)展都起到至關(guān)重要的作用。另外,在腦組織缺氧缺血損傷中,氧化應(yīng)激損傷也是一個(gè)核心病理過(guò)程,在機(jī)體抗氧化應(yīng)激防御機(jī)制中,Keap1-Nrf2/ARE信號(hào)通路是重要的化學(xué)通路之一,可以通過(guò)綜合的抗氧化、抗凋亡和抗炎特性起到保護(hù)細(xì)胞免受損害的作用,Nrf2調(diào)控下游血紅素加氧酶HO-1酶的表達(dá)。 腦損傷的細(xì)胞移植治療最早被認(rèn)為是一種神經(jīng)細(xì)胞的替代機(jī)制,目前認(rèn)為是一個(gè)多方面聯(lián)合的修復(fù)機(jī)制,移植細(xì)胞不僅可以直接替代受損細(xì)胞,而且可以通過(guò)促進(jìn)內(nèi)源性神經(jīng)干細(xì)胞、旁分泌營(yíng)養(yǎng)因子、改善局部血供等機(jī)制。因而本研究結(jié)合HIBD的發(fā)病機(jī)理,從抗炎性反應(yīng)和抗氧化應(yīng)激機(jī)制兩方面探討了PD-MSCs的作用機(jī)制。 研究目的 第一部分：從大鼠的生長(zhǎng)發(fā)育、神經(jīng)行為學(xué)、病理改變等方面評(píng)估PD-MSCs干預(yù)治療HIBD模型鼠的效果； 第二部分：從抑制炎性反應(yīng)、免疫調(diào)節(jié)、抗氧化應(yīng)激等方面深入探討PD-MSCs治療HIBD模型鼠的作用機(jī)制。 研究方法 1.從孕鼠胎盤(pán)分離、培養(yǎng)PD-MSCs,利用流式細(xì)胞術(shù)檢測(cè)細(xì)胞表型,利用體外誘導(dǎo)分化技術(shù)證明其多向分化能力,驗(yàn)證其是否符合間充質(zhì)干細(xì)胞的國(guó)際通用標(biāo)準(zhǔn)；然后利用GFP轉(zhuǎn)染標(biāo)記PD-MSCs,回輸新生鼠并觀察MSCs輸注后在體內(nèi)各主要組織器官的分布情況； 2.選取7日齡(postnatal day7, P7)健康Wistar大鼠,按照改良Rice法制作新生鼠HIBD模型。觀察大鼠行為學(xué)改變,采用Bederson評(píng)分進(jìn)行功能評(píng)分等級(jí),標(biāo)準(zhǔn)為：0分：沒(méi)有神經(jīng)損傷癥狀；1分：抓大鼠鼠尾提起,大鼠不能完全伸展對(duì)側(cè)前爪；2分：大鼠的前肢對(duì)對(duì)側(cè)推力的抵抗能力下降；3分：大鼠出現(xiàn)向?qū)?cè)轉(zhuǎn)圈現(xiàn)象。HIBD模型建立后48h,隨機(jī)分為對(duì)照組(Control)、 HIBD組(HIBD)、PD-MSCs治療組(HIBD+PD-MSCs)和成纖維細(xì)胞治療組(HIBD+Fibroblasts),在立體定向引導(dǎo)下,向大鼠腦組織內(nèi)勻速注射等量的PD-MSCs、成纖維細(xì)胞。 3.細(xì)胞治療后,觀察每只實(shí)驗(yàn)大鼠的生長(zhǎng)發(fā)育、皮膚外觀形態(tài)、體重的增長(zhǎng)情況及行為有無(wú)異常表現(xiàn)。 4.采用懸掛試驗(yàn)、滑棒試驗(yàn)評(píng)價(jià)大鼠的運(yùn)動(dòng)行為；水迷宮試驗(yàn)評(píng)價(jià)大鼠的認(rèn)知功能。懸掛試驗(yàn)、滑棒試驗(yàn)在P10、P16、P22和P28完成,Morris水迷宮試驗(yàn)在P24～P28完成。 5.細(xì)胞治療后5d, CD4+CD25+T淋巴細(xì)胞的檢測(cè)。 6.在大鼠缺氧缺血損傷后3h、6h、24h、3d、5d以及細(xì)胞治療后3d檢測(cè)了TNF-α、 IFN-γ、IL-10、IL-17mRNA的表達(dá)。 7.缺血缺氧損傷后6h、24h、48h、72h、5d以及細(xì)胞治療后5d時(shí)檢測(cè)了HO-1、 Nrf2mRNA的表達(dá)。 8.細(xì)胞治療后5d,采用Western blot法分別對(duì)脾臟組織中Foxp3和海馬組織中HO-1、Nrf2進(jìn)行檢測(cè)。 9.細(xì)胞治療后3d,應(yīng)用ELISA法檢測(cè)外周血清中TNF-α、IFN-γ、IL-10、IL-17的水平。 10.缺血缺氧損傷后6h,24h,48h,72h以及細(xì)胞治療后5d,檢測(cè)海馬組織中MDA含量。 11.P28運(yùn)動(dòng)實(shí)驗(yàn)完成后,進(jìn)行HE染色和尼氏染色并行病理分析。 結(jié)果 1.從孕鼠胎盤(pán)可以分離、培養(yǎng)出MSCs,利用流式細(xì)胞術(shù)和體外細(xì)胞誘導(dǎo)分化技術(shù),證明所獲細(xì)胞符合間充質(zhì)干細(xì)胞鑒定標(biāo)準(zhǔn),驗(yàn)證了從大鼠胎盤(pán)獲得MSCs是完全可行的。 2.新生鼠在缺氧開(kāi)始時(shí)出現(xiàn)煩躁、紫紺,其后逐漸轉(zhuǎn)為抑制,甚至出現(xiàn)抽搐。HIBD組與對(duì)照組相比體重明顯減輕。懸掛試驗(yàn)、滑桿試驗(yàn)中,HIBD組大鼠與正常對(duì)照組比較表現(xiàn)出明顯的運(yùn)動(dòng)機(jī)能落后,Morris水迷宮定位航行試驗(yàn)中,HIBD組與對(duì)照組相比逃避潛伏期明顯延長(zhǎng),在第6天的空間探索試驗(yàn)中,HIBD組穿過(guò)原平臺(tái)的頻率明顯降低。腦組織大體可見(jiàn)腫脹、蒼白；后期有萎縮、梗死、液化。HE染色鏡下見(jiàn)損傷側(cè)皮層、海馬神經(jīng)細(xì)胞、小腦浦肯野細(xì)胞排列紊亂、變性、壞死。尼氏染色尼氏小體消退,出現(xiàn)神經(jīng)元嗜酸性變、膠質(zhì)細(xì)胞噬細(xì)胞現(xiàn)象。從生長(zhǎng)發(fā)育、運(yùn)動(dòng)功能評(píng)估、病理改變等多方面判斷HIBD模型是成功的。 3.PD-MSCs腦內(nèi)遷移：移植后6小時(shí),GFP陽(yáng)性PD-MSCs被發(fā)現(xiàn)主要是在注射部位。移植后6天,綠色熒光信號(hào)明顯減弱。GFP陽(yáng)性細(xì)胞在接受注射的大鼠缺血側(cè),而對(duì)側(cè)皮層沒(méi)有發(fā)現(xiàn)GFP陽(yáng)性細(xì)胞。PD-MSCs主要在注射針道和／或注射部位被發(fā)現(xiàn)。然后大量細(xì)胞遷出注射部位、擴(kuò)散到整個(gè)皮層和腦室周圍區(qū)域。 4.流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示,HIBD組CD4+CD25+T細(xì)胞為11.4%,比正常對(duì)照組(8.69%)略有升高,細(xì)胞治療后,成纖維細(xì)胞治療組CD4+CD25+T細(xì)胞15.1%, PD-MSCs治療組CD4+CD25+T細(xì)胞則顯著升高,達(dá)18.0%。Western Blot檢測(cè)大鼠脾臟Foxp3的蛋白含量,結(jié)果發(fā)現(xiàn)HIBD組脾臟Foxp3蛋白含量雖高于正常對(duì)照組,但無(wú)顯著性差異,經(jīng)過(guò)PD-MSCs治療后,大鼠脾臟Foxp3的蛋白表達(dá)明顯增多,與HIBD組比較具有統(tǒng)計(jì)學(xué)意義。 5.缺氧缺血損傷后,腦組織中IFN-γ、TNF-α、IL-17和IL-10mRNA的動(dòng)態(tài)變化結(jié)果：HIBD組TNF-α,IFN-γ和IL-17mRNA表達(dá)大幅上調(diào),TNF-a mRNA在損傷后3h表達(dá)開(kāi)始增加,在24h時(shí)表達(dá)大幅度升高,明顯高于對(duì)照組。IFN-γ mRNA、IL-17mRNA表達(dá)升高時(shí)間較TNF-α有所延遲,其二者的表達(dá)高峰在HI損傷后3d。IL-10mRNA的表達(dá)在6h開(kāi)始上調(diào),在3d后達(dá)高峰。PD-MSCs治療前后IFN-γ、TNF-α、IL-17和IL-10細(xì)胞因子水平比較：RT-PCR法檢測(cè)PD-MSCs治療組TNF-α, IFN-γ和IL-17mRNA表達(dá)明顯低于HIBD組和成纖維細(xì)胞組,在HIBD組、成纖維細(xì)胞組和PD-MSCs治療組三個(gè)實(shí)驗(yàn)組,IL-10mRNA的表達(dá)與對(duì)照組比較均明顯上調(diào),在PD-MSCs治療組升高尤為明顯,明顯高于HIBD組和成纖維細(xì)胞組。 6.ELISA法檢測(cè)結(jié)果：HIBD組血清IFN-γ TNF-α、IL-17和IL-10水平均高于正常對(duì)照組,PD-MSCs治療后,大鼠血清IFN-γ、TNF-α和IL-17水平較HIBD組、成纖維細(xì)胞治療組均明顯有所下降,而IL-10水平則顯著有所升高,HIBD組與成纖維細(xì)胞治療組之間IFN-γ、TNF-α、IL-17和IL-10水平?jīng)]有顯著差異。 7. HIBD中HO-1、Nrf2的動(dòng)態(tài)變化：HIBD組HO-1和Nrf2mRNA的表達(dá)與對(duì)照組相比較在6h開(kāi)始上調(diào)、48h達(dá)高峰,其后HO-1和Nrf2mRNA的表達(dá)雖然逐漸下降,但在72h和5d仍保持在較高的水平。PD-MSCs治療前后HO-1、Nrf2表達(dá)的比較：RT-PCR法檢測(cè)HIBD組、成纖維細(xì)胞組和PD-MSCs治療組三個(gè)實(shí)驗(yàn)組的HO-1和Nrf2mRNA的表達(dá)均高于正常對(duì)照組,而PD-MSCs治療組則顯著高于HIBD組和成纖維細(xì)胞組。Western blot分析表明HO-1和Nrf2在HIBD組、PD-MSCs治療組和成纖維細(xì)胞治療組均比對(duì)照組明顯增多,其中PD-MSCs治療組較HIBD組、成纖維細(xì)胞治療組均明顯增高。 8.MDA水平動(dòng)態(tài)變化及治療前后對(duì)比：缺氧缺血性損傷后,腦組織MDA水平在HI后6h時(shí)開(kāi)始顯著升高,72h達(dá)峰值水平,隨之MDA水平開(kāi)始逐漸降低,5d時(shí)MDA水平雖顯著降低,但仍然高于正常水平。PD-MSCs治療組MDA水平與缺氧缺血組、成纖維細(xì)胞治療組相比明顯降低,具有顯著性差異。 結(jié)論 1.從行為學(xué)、神經(jīng)運(yùn)動(dòng)功能、病理改變等方面證實(shí)改良Rice法建造的HIBD模型是可行的、成功的。 2.組織塊貼壁法可以成功培養(yǎng)大鼠PD-MSCs。 3. PD-MSCs干預(yù)不但能改善HIBD大鼠近期的神經(jīng)運(yùn)動(dòng)功能,而且能改善HIBD大鼠的遠(yuǎn)期學(xué)習(xí)記憶功能。 4. HIBD的發(fā)病機(jī)制之一是：促炎性細(xì)胞因子IFN-γ、TNF-α、IL-17和抗炎性細(xì)胞因子IL-10之間的失衡,同時(shí)存在CD4+CD25+Treg細(xì)胞功能紊亂。 5. HIBD的發(fā)病機(jī)制之一是：抗氧化(Nrf2/HO-1)／氧化(MDA)水平之間的失衡。 6. PD-MSCs治療HIBD有效的機(jī)制之一可能是通過(guò)調(diào)節(jié)Th17/Treg細(xì)胞失衡。 7.PD-MSCs治療HIBD的作用靶點(diǎn)之一可能是上調(diào)Keap1-Nrf2/ARE/HO-1通路。
[Abstract]:Research background
Hypoxic-ischemic brain damage (HIBD) is a neonatal brain injury caused by perinatal asphyxia, hypoxia, and ischemia. Perinatal asphyxia leads to moderate or severe hypoxic-ischemic encephalopathy in 3 to 5/1000 living babies, which often leads to neonatal death and some neurological dysfunction, such as brain. Paralysis, epilepsy and mental retardation. Among them, cerebral palsy is the most important motor disability disease in childhood. It can cause the disabled children for life, the high incidence of HIBD, the rate of disability to the children himself, family and society have caused great mental and economic burden.
The pathogenesis of HIBD is a very complicated pathological process, which is the result of a series of chain reactions involved in a variety of mechanisms. The exact pathogenesis is not clear at present. At present, the treatment of neonatal HIBD is mostly used in the treatment of hyperbaric oxygen, neurotrophic drugs, physical rehabilitation and so on, which are relatively light to the degree of injury. It has a certain effect, but the central nervous dysfunction caused by moderate to severe encephalopathy is difficult to work. Therefore, it is of great clinical significance to seek an effective treatment to reduce the mortality and disability rate of children with HIBD.
Recent studies have shown that mesenchymal stem cells (MSCs) has made great progress in the animal and preclinical trials of HIBD. At present, there are many studies on bone marrow MSCs, but BMSCs has less stem cell content, weak proliferation and differentiation potential, invasive operation, and increased probability of virus infection. Limitations, which make its clinical application and Promotion Limited. This study confirms that the placenta can be separated from the pregnant mouse, and the placental source MSCs (placenta-derived mesenchymal stem cells, PD-MSCs) is cultured. It is confirmed that it is suitable for the identification of mesenchymal stem cells. It is proved that it is completely feasible to obtain MSCs from the rat placenta, and PD-MSCs is superior to it for its own advantages. In the future, it may become an ideal seed cell with great potential for application.
The pathogenesis of HIBD involves inflammatory reaction, oxidative stress injury, and brain tissue energy metabolism disorder. The inflammatory response after ischemia has become the focus of research. Inflammatory cells can infiltrate into the brain parenchyma and release a variety of neurotoxic substances to cause inflammatory cascade reaction. Multiple cytokines are involved, and T cells 17 (T helper) are assisted. Cell17, Th17) and regulatory T cells (regulatory T cells, Treg) also play an important role in the development of HIBD. In addition, oxidative stress damage is also a core pathological process in the brain tissue hypoxic and ischemic injury. In the system of anti oxidative stress defense mechanism, the Keap1-Nrf2/ARE signaling pathway is an important chemical pathway. First, Nrf2 can regulate the expression of heme oxygenase HO-1 enzyme through comprehensive antioxidant, anti apoptotic and anti-inflammatory properties.
Cell transplantation for brain injury is considered as an alternative mechanism for neural cells. It is considered to be a multifaceted joint repair mechanism. Transplanted cells can not only directly replace damaged cells, but also improve local blood supply by promoting endogenous neural stem cells, paracrine nutrient factors and improving local blood supply. Combined with the pathogenesis of HIBD, the mechanism of PD-MSCs was discussed from two aspects: anti-inflammatory reaction and anti oxidative stress mechanism.
research objective
Part one: To evaluate the effect of PD-MSCs intervention on HIBD model rats from the aspects of growth and development, neurobehavioral and pathological changes.
The second part: from the aspects of inhibiting inflammatory reaction, immunoregulation and anti oxidative stress, we will further explore the mechanism of PD-MSCs in the treatment of HIBD mice.
research method
1. isolated from the placenta of pregnant rats, cultured PD-MSCs, using flow cytometry to detect cell phenotype, using in vitro differentiation technique to prove its multidirectional differentiation ability, verify whether it conforms to the international standard of mesenchymal stem cells, and then transfection of PD-MSCs with GFP transfection, retransfuse new mice and observe the main organs after MSCs infusion in the body. The distribution of officials;
2. the healthy Wistar rats of 7 days of age (postnatal Day7, P7) were used to make the HIBD model of newborn rats according to the improved Rice method. The behavioral changes were observed by the Bederson score. The standard was 0 points: no nerve injury symptoms; 1: grabbing the rat tail, the rats could not completely extend the contralateral front claw; 2 points: rats: rats: 2 points: Rats The resistance of the forelimb to the contralateral thrust decreased; 3: after the.HIBD model was established to the contralateral circle, the rats were randomly divided into the control group (Control), the HIBD group (HIBD), the PD-MSCs treatment group (HIBD+PD-MSCs) and the fibroblast treatment group (HIBD+Fibroblasts). Under the stereotactic guidance, the constant injection of the same amount to the rat brain was equal. PD-MSCs, fibroblasts.
3. after treatment, the growth, skin appearance, weight gain and behavior of each experimental rat were observed.
4. suspension test was used to evaluate the exercise behavior of rats. The water maze test was used to evaluate the cognitive function of rats. Suspension test was performed and the slide test was completed in P10, P16, P22 and P28, and the Morris water maze test was completed in P24 to P28.
5. detection of 5D and CD4+CD25+T lymphocytes after cell therapy.
6. after 3h, 6h, 24h, 3D, 5D and 3D treatment, the expressions of TNF-, IFN-, IL-10 and IL-17mRNA were detected in rats after hypoxia ischemia injury.
7. after ischemia and hypoxia, 6h, 24h, 48h, 72h, 5D and 5D expression in cells were detected for HO-1 and Nrf2mRNA expression.
8. after 5D, Western and blot were used to detect Foxp3 and HO-1 and Nrf2 in the hippocampus respectively.
9. after 3D, the levels of TNF-, IFN-, IL-10 and IL-17 in peripheral serum were detected by ELISA.
10. after ischemia and hypoxia, 6h, 24h, 48h, 72h and 5D after cell therapy were used to detect the content of MDA in hippocampus tissue.
After 11.P28 exercise test, HE staining and Nissl staining were performed and pathological analysis was performed.
Result
1. from the placenta of pregnant mice, MSCs was isolated and cultured. Using flow cytometry and in vitro cell differentiation, it was proved that the obtained cells met the criteria for identification of mesenchymal stem cells. It was proved that it was completely feasible to obtain MSCs from the rat placenta.
2. the newborn rats appeared irritable and cyanotic at the start of anoxia, and then gradually turned to inhibition, and even the group of convulsions.HIBD group had a significant reduction in weight compared with the control group. In the suspension test, the HIBD group showed obvious motor function lag compared with the normal control group. In the Morris water maze navigation test, the HIBD group and the control group In the sixth day space exploration test, the frequency of the HIBD group passed the original platform significantly decreased in the space exploration test. The brain tissue was generally swollen and pale. There was atrophy, infarction, and liquefied.HE stained lens, the injured lateral cortex, the hippocampal neurons, the cerebellum puken field cells arranged disorder, denaturation, necrosis. Nissl staining Nissl's small The body subsided, the neuron eosinophilic change, the glial cell phage phenomenon. It was successful to judge the HIBD model from many aspects, such as growth and development, motor function evaluation, pathological changes and so on.
3.PD-MSCs intracerebral migration: 6 hours after transplantation, GFP positive PD-MSCs was found mainly at the injection site. 6 days after the transplantation, the green fluorescent signal significantly weakened the.GFP positive cells in the ischemic side of the injected rat, while the GFP positive cells in the lateral cortex were not found to be found mainly in the injection and / or injection sites. The cells moved out of the injection site and spread throughout the cortex and periventricular area.
The results of 4. flow cytometry showed that CD4+CD25+T cells in group HIBD were 11.4%, slightly higher than that in normal control group (8.69%). After cell therapy, CD4+CD25+T cells in fibroblast treatment group were 15.1%, CD4+CD25+T cells in PD-MSCs treatment group increased significantly, and 18.0%.Western Blot was used to detect the protein content of spleen Foxp3 in rats, and the results were found in the HIBD group of spleen. Although the content of Foxp3 protein was higher than that of the normal control group, there was no significant difference. After PD-MSCs treatment, the expression of Foxp3 protein in the spleen increased significantly, and was statistically significant compared with that of the HIBD group.
5. the dynamic changes of IFN- gamma, TNF- a, IL-17 and IL-10mRNA in the brain tissue after the hypoxic and ischemic injury: the expression of TNF- alpha, IFN- gamma and IL-17mRNA in HIBD group increased significantly. The 3H expression of TNF-a mRNA began to increase after the injury, and the expression in 24h was significantly higher than that of the control group. The expression of 3d.IL-10mRNA was up to rise after HI injury, and the level of IFN- gamma, TNF- a, IL-17 and IL-10 cytokine before and after the peak.PD-MSCs treatment after 3D was compared: RT-PCR method was used to detect TNF- alpha in PD-MSCs treatment group, and the expression was significantly lower than that of the group and fibroblast group, and the fibroblasts were in the group of fibroblasts. In the three experimental groups of the group and the PD-MSCs treatment group, the expression of IL-10mRNA was significantly up-regulated compared with the control group, especially in the PD-MSCs treatment group, which was significantly higher than that in the HIBD group and the fibroblast group.
The results of 6.ELISA assay: serum IFN- gamma TNF- alpha, IL-17 and IL-10 levels in group HIBD were higher than that in normal control group. After PD-MSCs treatment, serum IFN- gamma, TNF- alpha and IL-17 levels were significantly lower than those in the HIBD group, while the IL-10 level was significantly higher. There was no significant difference in IL-17 and IL-10 levels.
The dynamic changes of HO-1 and Nrf2 in 7. HIBD: the expression of HO-1 and Nrf2mRNA in group HIBD compared with the control group was up up, the peak of 48h reached, and the expression of HO-1 and Nrf2mRNA decreased gradually, but the comparison between 72h and 5D still remained at a higher level. The expression of HO-1 and Nrf2mRNA in the three experimental groups of the PD-MSCs treatment group was higher than that in the normal control group, while the PD-MSCs treatment group was significantly higher than the HIBD group and the fibroblast group.Western blot analysis showed that HO-1 and Nrf2 were in the HIBD group, the PD-MSCs treatment group and the fibroblast treatment group were all significantly higher than those in the control group, and the PD-MSCs treatment group was more than that of the control group. The treatment group of fibroblast increased significantly.
8.MDA level dynamic changes and before and after treatment: after hypoxic ischemic injury, the brain tissue MDA level began to increase significantly at 6h after HI, 72h reached the peak level, and then MDA level began to decrease gradually, while MDA level decreased significantly in 5D, but still higher than normal level.PD-MSCs treatment group MDA level and hypoxia ischemia group, fibroblast treatment. The group was significantly lower than the group, and there was a significant difference.
conclusion
1. it is proved that the improved Rice method is feasible and successful in terms of ethology, neuromotor function and pathological changes. The HIBD model constructed by the improved method is feasible and successful.
2. tissue block adherence method can successfully cultivate rat PD-MSCs..
3. PD-MSCs intervention can not only improve the short-term motor function of HIBD rats, but also improve the long-term learning and memory function of HIBD rats.
One of the pathogenesis of 4. HIBD is the imbalance between proinflammatory cytokines IFN- gamma, TNF- alpha, IL-17 and anti-inflammatory cytokine IL-10, and there is a dysfunction of CD4+CD25+Treg cells.
One of the pathogenesis of 5. HIBD is the imbalance between antioxidant (Nrf2/HO-1) and oxidative (MDA) levels.
One of the effective mechanisms of 6. PD-MSCs treatment of HIBD may be to regulate Th17/Treg cell imbalance.
One of the targets of 7.PD-MSCs therapy for HIBD may be up regulation of Keap1-Nrf2/ARE/HO-1 pathway.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742

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