本文選題：脂肪間充質(zhì)干細(xì)胞 + 心搏驟停　； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：近些年來,心肺復(fù)蘇的成功率逐漸提高,但由于神經(jīng)細(xì)胞對缺血缺氧敏感而且不能再生,大部分患者復(fù)蘇后出現(xiàn)不可逆的神經(jīng)損傷,導(dǎo)致失語、偏癱、植物人等后遺癥甚至導(dǎo)致死亡。盡管采取了亞低溫治療、藥物治療等綜合措施治療心搏驟停后的神經(jīng)損傷,但是心搏驟停患者的出院生存率仍不高,在中國心搏驟�；颊呱窠�(jīng)功能良好的出院率僅1%左右。如何減輕神經(jīng)損傷、促進(jìn)神經(jīng)修復(fù)已成為現(xiàn)代醫(yī)學(xué)的難題,在此我們嘗試一種新措施治療心搏驟停引起的神經(jīng)損傷。間充質(zhì)干細(xì)胞具有高度自我更新能力,可以多次傳代、大量擴(kuò)增;同時(shí)還具有多向分化潛能,在一定的誘導(dǎo)條件下能分化為脂肪細(xì)胞、成骨細(xì)胞、軟骨細(xì)胞、神經(jīng)細(xì)胞等。ADSCs是來源于脂肪組織的MSCs,其儲(chǔ)備豐富,取材容易,其增殖分化能力高度穩(wěn)定,不會(huì)隨供者年齡產(chǎn)生變化。目前研究發(fā)現(xiàn)ADSCs在小動(dòng)物腦梗塞模型中取得了良好效果,ADSCs移植能減少腦梗面積,減輕局灶性神經(jīng)損傷,改善神經(jīng)功能預(yù)后。而腦梗塞為局部性病灶,與心搏驟停引起的全腦范圍的缺氧性腦病,在病因、病理機(jī)制、臨床表現(xiàn)上都有所不同,ADSCs對于心搏驟停引起的全腦范圍的缺氧性腦病的治療效果及治療機(jī)制需要進(jìn)一步驗(yàn)證。本實(shí)驗(yàn)研究異種移植ADSCs對心搏驟停后缺氧性腦病的治療效果,并探討其作用機(jī)制,為臨床治療提供理論支持。主要內(nèi)容包括:人ADSCs的培養(yǎng),從細(xì)胞形態(tài)、細(xì)胞表型、多向分化能力等方面進(jìn)行鑒定;采用窒息法建立大鼠心肺復(fù)蘇模型并移植人ADSCs,觀察ADSCs對心搏驟停后缺氧性腦病的治療效果,海馬組織內(nèi)BDNF、IL-6的表達(dá)情況。第一部分ADSCs培養(yǎng)及鑒定研究目的從人脂肪組織中分離、培養(yǎng)ADSCs,并從細(xì)胞形態(tài)、細(xì)胞表型、分化能力(成骨分化、成軟骨分化、成脂肪分化)等方面進(jìn)行鑒定,為下一步動(dòng)物實(shí)驗(yàn)奠定基礎(chǔ)。材料和方法從健康供者腹部抽取脂肪組織,消化、分離出血管基質(zhì)部分,在37℃、5%CO2培養(yǎng)箱中使用DMEM/F12+10%胎牛血清培養(yǎng)基中貼壁培養(yǎng),逐漸純化為ADSCs,每3天換液1次,待細(xì)胞70%~90%融合時(shí)進(jìn)行傳代,培養(yǎng)過程中觀察細(xì)胞形態(tài)。取第3代ADSCs采用流式細(xì)胞術(shù)檢測細(xì)胞表面抗原CD73、CD90、CD105、CD34、CD45、CD11b、CD19、HLA-DR的表達(dá)情況。使用成骨分化試劑盒誘導(dǎo)成骨分化,第28天茜素紅染色法、磷酸苯二鈉基質(zhì)(NBT/BCIP)染色法檢測成骨分化情況;使用成軟骨分化試劑盒誘導(dǎo)成軟骨分化,第7天阿利辛藍(lán)染色檢測成軟骨分化情況;使用成脂分化試劑盒誘導(dǎo)成脂分化,第21天油紅O染色法檢測成脂分化情況。結(jié)果原代培養(yǎng)24小時(shí)后,鏡下可見短梭形、三角形等多種形態(tài)的貼壁細(xì)胞,隨著時(shí)間增長,梭形細(xì)胞逐漸增多,呈長梭形外觀,細(xì)胞成集落樣生長,在6~8天后達(dá)到80%左右融合。傳代后始終保持著穩(wěn)定的增殖速度。adscs表面抗原cd73、cd90、cd105陽性表達(dá)率高,分別為99.99%、99.99%、96.88%,同時(shí)陰性表達(dá)cd34、cd45、cd11b、cd19、hla-dr。成骨誘導(dǎo)后細(xì)胞生長旺盛,細(xì)胞形態(tài)由長梭形逐漸變?yōu)槎趟笮?7天后可見結(jié)節(jié)樣結(jié)構(gòu),28天后礦化結(jié)節(jié)形成明顯,礦化結(jié)節(jié)被茜素紅染色法染成紅色結(jié)節(jié),胞質(zhì)因含有堿性磷酸被nbt/bcip染色法染成深藍(lán)色。成軟骨誘導(dǎo)后,細(xì)胞形態(tài)由長梭形逐漸回縮變圓,7天后用阿利辛藍(lán)染色檢測軟骨細(xì)胞基質(zhì)中糖胺多糖合成情況,胞質(zhì)被染成藍(lán)色。成脂誘導(dǎo)后,細(xì)胞形態(tài)由長梭形逐漸變?yōu)閳A形,胞漿內(nèi)可見小脂滴,脂滴數(shù)量逐漸增加并相互融合,14天后油紅o染色可見細(xì)胞內(nèi)脂滴被染成紅色。結(jié)論從人脂肪組織中分離并培養(yǎng)出穩(wěn)定的adscs,從細(xì)胞形態(tài)、細(xì)胞表面抗原、多向分化能力3個(gè)方面對細(xì)胞進(jìn)行鑒定,符合mscs的鑒定標(biāo)準(zhǔn)。第二部分觀察adscs移植對心肺復(fù)蘇后缺氧性腦病的治療效果及保護(hù)機(jī)制研究研究目的觀察異種adscs移植對大鼠心搏驟停后神經(jīng)功能、神經(jīng)元凋亡、血清s100β的影響,海馬組織bdnf、il-6的表達(dá)情況材料和方法54只大鼠隨機(jī)分為3組(sham組、ca組、adscs組),每組18只。sham組只進(jìn)行外科操作,不進(jìn)行心搏驟停及心肺復(fù)蘇操作。ca組使用氣管夾閉法誘導(dǎo)窒息性心搏驟停模型,心搏停止6分鐘,復(fù)蘇后1小時(shí)靜脈注射1mlpbs。adscs組建立窒息性心搏驟停模型,心搏停止6分鐘,復(fù)蘇后1小時(shí)靜脈注射人adscs1ml(細(xì)胞計(jì)數(shù)5×106)。操作完成后24小時(shí)、72小時(shí)、168小時(shí),每組隨機(jī)選取6只大鼠進(jìn)行檢測。使用神經(jīng)功能缺損評分評價(jià)神經(jīng)功能,elisa法檢測血清s100β濃度,he染色觀察海馬組織病理變化,tunel染色檢測海馬神經(jīng)元凋亡率,werstern-blot檢測海馬bdnf、il-6蛋白水平。結(jié)果1.心搏驟停前采集到的大鼠體重、平均動(dòng)脈壓、心率,比較差異無統(tǒng)計(jì)學(xué)意義(p0.05),心肺復(fù)蘇期間窒息時(shí)間、按壓時(shí)間及復(fù)蘇后1小時(shí)map比較差異無統(tǒng)計(jì)學(xué)意義(p0.05)。2.神經(jīng)功能缺損評分在24小時(shí)、72小時(shí)、168小時(shí),sham組大鼠評分高于adscs組和ca組,差異具有統(tǒng)計(jì)學(xué)意義(p0.05),adscs組均高于ca組,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。3.病理變化sham組大鼠結(jié)果正常。CA組大鼠海馬區(qū)錐體細(xì)胞減少,錐體細(xì)胞稀疏,大量小膠質(zhì)細(xì)胞增生。ADSCs組大鼠海馬區(qū)少量細(xì)胞核皺縮改變,錐體細(xì)胞排列基本正常。4.TUNEL染色在3個(gè)時(shí)間點(diǎn),sham組神經(jīng)元凋亡率明顯低于CA組和ADSCs組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),而ADSCs組神經(jīng)元凋亡率均低于CA組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。5.血清S100β濃度在3個(gè)時(shí)間點(diǎn),sham組血清S100β濃度均低于CA組和ADSCs組差異具有統(tǒng)計(jì)學(xué)意義(P0.01),而ADSCs組S100β濃度低于CA組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。6.海馬IL-6在3個(gè)時(shí)間點(diǎn),sham組IL-6表達(dá)水平低于CA組和ADSCs組,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。在24小時(shí)、72小時(shí),ADSCs組IL-6高于CA組,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。復(fù)蘇后168小時(shí),ADSCs組和CA組IL-6比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。7.海馬BDNF在復(fù)蘇后24小時(shí)和72小時(shí),ADSCs組和CA組BDNF蛋白水平高于sham組(P0.01),而ADSCs組BDNF蛋白高于CA組,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。在復(fù)蘇后168小時(shí),3組BDNF蛋白表達(dá)強(qiáng)度接近,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論心搏驟停能引起神經(jīng)凋亡、神經(jīng)功能障礙,ADSCs具有神經(jīng)保護(hù)作用,能抑制神經(jīng)細(xì)胞凋亡,改善神經(jīng)功能預(yù)后。ADSCs可能是通過上調(diào)BDNF、IL-6表達(dá)發(fā)揮神經(jīng)保護(hù)作用。
[Abstract]:In recent years, the success rate of cardiopulmonary resuscitation has gradually increased, but because nerve cells are sensitive to ischemia and hypoxia and can not be regenerated, most patients have irreversible nerve damage after resuscitation, resulting in aphasia, hemiplegia, vegetative sequelae and even death. Although mild hypothermia treatment, drug treatment and other comprehensive measures for the treatment of heart beat The survival rate of patients with cardiac arrest is still not high, and the discharge rate is only about 1% in patients with cardiac arrest. How to reduce nerve injury and promote nerve repair has become a difficult problem in modern medicine. Here we try a new measure to treat the nerve injury caused by cardiac arrest. Stem cells have the ability of high self renewal, which can be passaged and proliferate many times. At the same time, it also has the potential to differentiate into adipocytes under certain induction conditions. The.ADSCs of osteoblasts, chondrocytes and nerve cells is the MSCs derived from adipose tissue, which is rich in reserve and easy to obtain, and its proliferation and differentiation ability is highly stable. The present study has found that ADSCs has achieved good results in the model of cerebral infarction in small animals. ADSCs transplantation can reduce the area of cerebral infarction, reduce focal nerve damage and improve the prognosis of nerve function. Cerebral infarction is a local focus, and the whole brain area of the brain is induced by cardiac arrest, in the etiology and pathology. The mechanism and clinical manifestations are different. The therapeutic effect and treatment mechanism of ADSCs for the whole brain range of hypoxic encephalopathy caused by cardiac arrest are further verified. This experiment studies the therapeutic effect of xenotransplantation of ADSCs on hypoxic encephalopathy after cardiac arrest, and discusses its mechanism for clinical treatment. The contents include: the cultivation of human ADSCs, the identification of cell morphology, cell phenotype, and multidirectional differentiation, and the establishment of rat cardiopulmonary resuscitation model by asphyxia method and transplantation of human ADSCs, to observe the therapeutic effect of ADSCs on hypoxic encephalopathy after cardiac arrest, the expression of BDNF and IL-6 in the hippocampus. The first part of ADSCs culture and Identification Research The purpose of this study was to isolate and cultivate ADSCs from human adipose tissue, and to identify the cell morphology, cell phenotype, differentiation capacity (osteogenesis differentiation, chondrodifferentiation, adipose differentiation) and other aspects, which lay the foundation for the next animal experiment. Materials and methods were extracted from the abdominal fat tissue of the healthy donor, digested and separated from the vascular matrix, at 37, 5%. The CO2 culture box was cultured in the culture medium of DMEM/F12+10% fetal bovine serum, which was gradually purified to ADSCs and changed 1 times every 3 days. The cell morphology was observed during the cell 70%~90% fusion. The cell surface antigen CD73, CD90, CD105, CD34, CD45, CD11b, CD19, and expression were detected by flow cytometry for the third generation of ADSCs. Osteogenesis differentiation was induced by osteogenic differentiation kit, twenty-eighth days alizarin red staining, benzyl benzene two matrix (NBT/BCIP) staining method was used to detect osteogenic differentiation, chondrodifferentiation was induced by chondrogenic differentiation kit, and chondrodifferentiation was detected by alizocin blue staining at seventh days, fat differentiation was induced by fat differentiation kit and twenty-first days. Oil red O staining was used to detect the formation of lipid differentiation. Results after 24 hours of primary culture, the cells with short shuttle shape, triangle and other morphologic cells were seen under the microscope. The spindle cells gradually increased with time, and the cells grew in a long shuttle shape. The cells became colony like growth and reached about 80% after 6~8 days. The positive expression rate of.Adscs surface antigen CD73, CD90, CD105 was high, 99.99%, 99.99%, 96.88% respectively. Meanwhile, the negative expression of CD34, CD45, CD11b, CD19, and HLA-DR. became strong, and the cell morphology changed from long spindle shape to short shuttle form, and nodular structure was visible after 7 days. 28 days after the mineralization, the mineralized nodules were formed and the mineralized nodules were alizarin red. The staining method was dyed red nodules, cytoplasm was dyed deep blue by nbt/bcip staining with alkaline phosphoric acid. After induction of cartilage, the cell morphology was gradually retracted from long spindle shape, and 7 days later, the synthesis of glycosaminoglycan in chondrocyte matrix was detected by alisocen blue, and the cytoplasm was stained blue. The gradient is round, small fat droplets are visible in the cytoplasm, the number of lipid droplets gradually increases and fuses each other. 14 days after the oil red O staining, the lipid droplets in the cells are dyed red. Conclusion a stable ADSCs is separated and cultured from the human adipose tissue, and the cells are identified from the cell morphology, the cell surface antigen and the multi-directional differentiation ability, which conforms to the MSCs. The second part observed the therapeutic effect and protective mechanism of ADSCs transplantation on hypoxic encephalopathy after cardiopulmonary resuscitation. Objective To observe the effects of Xenotransplantation on nerve function, neuron apoptosis, serum S100 beta, the expression of BDNF, IL-6 in hippocampus and 54 rats randomly divided into 3 groups. (Group sham, group Ca, group ADSCs), each group of 18.Sham groups only performed surgical operation, without cardiac arrest and cardiopulmonary resuscitation, group.Ca used tracheal clamping to induce asphyxiating cardiac arrest model, cardiac arrest for 6 minutes, and 1 hours after resuscitation by intravenous injection of 1mlpbs.adscs group to establish asphyxiating cardiac arrest model, cardiac arrest for 6 minutes, and 1 small after resuscitation. Intravenous injection of human adscs1ml (cell count 5 x 106). After 24 hours, 72 hours, 168 hours after completion of the operation, 6 rats were randomly selected for each group. Nerve function defect score was used to evaluate the nerve function, ELISA method was used to detect the serum S100 beta concentration, he staining was used to observe the pathological changes of hippocampus, and TUNEL staining was used to detect the apoptosis rate of hippocampal neurons, werst Ern-blot detected the BDNF and IL-6 protein levels in the hippocampus. Results there was no statistically significant difference in weight, mean arterial pressure and heart rate before 1. cardiac arrest (P0.05). The time of asphyxia during cardiopulmonary resuscitation, compression time and 1 hour map after resuscitation were not statistically significant (P0.05).2. neural function defect score was 24 hours, 72 small At 168 hours, the score of rats in group sham was higher than that in group ADSCs and group Ca, the difference was statistically significant (P0.05), the group ADSCs was higher than that of the CA Group, the difference was statistically significant (P0.05).3. pathological changes in group sham rats, the result of the normal group of the rat hippocampus pyramidal cells decreased, the pyramidal cells were sparse, a large number of microglia proliferated the hippocampus of the.ADSCs group. A small amount of nuclear crinkle changed, the pyramidal cells were arranged basically with normal.4.TUNEL staining at 3 time points. The apoptosis rate of sham group was significantly lower than that of CA group and ADSCs group. The difference was statistically significant (P0.05), but the apoptosis rate of ADSCs group was lower than that in CA group, and the difference has statistical significance (P0.05).5. serum S100 beta concentration at 3 time points, sham The serum S100 beta concentration was lower than that of the CA group and the ADSCs group (P0.01), while the S100 beta concentration in the ADSCs group was lower than the CA group. The difference was statistically significant (P0.05).6. hippocampal IL-6 at 3 time points. The IL-6 expression level of sham group was lower than that of the CA group. In group CA, the difference was statistically significant (P0.01). 168 hours after resuscitation, there was no significant difference in IL-6 between group ADSCs and CA group (P0.05),.7. hippocampal BDNF was 24 hours and 72 hours after resuscitation, and BDNF protein level in ADSCs group and CA group was higher than that in sham group. 68 hours, the 3 groups of BDNF protein expression intensity close, the difference is not statistically significant (P0.05). Conclusion cardiac arrest can cause nerve apoptosis, neural dysfunction, ADSCs have neuroprotective effect, can inhibit the apoptosis of nerve cells, improve the prognosis of neural function.ADSCs may be through the upper modulation of BDNF, IL-6 expression to play neuroprotective effect.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R459.7

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