【作者】 李鹏；

【导师】 关方霞；

【作者基本信息】 郑州大学 ， 细胞生物学， 2018， 硕士

【摘要】 创伤性脑损伤（Traumatic brain injury,TBI）是一种常见的由于外力引起脑部神经功能障碍和神经细胞死亡的损伤性疾病,能够导致严重的生理、心理以及认知功能方面障碍。为了有效治疗TBI,迫切需要一种新型的治疗方法。干细胞与再生医学的出现为TBI治疗提供新的思路。研究发现,干细胞移植可以通过旁分泌和细胞替代途径对TBI发挥保护作用。人脐带间充质干细胞（Human umbilical cord mesenchymal stem cells,hUC-MSCs）具有更新速度快、低免疫原性和畸胎瘤形成风险低等特点,具有潜在的应用价值。TBI损伤之后由于组织缺血和炎症反应会导致产生大量的活性氧,引起组织病理和细胞死亡,阻碍移植后细胞的迁移和存活。MG53蛋白（Mitsugumin53 protein,MG53）是TRIM（Tripartite motif,TRIM）家族蛋白成员之一,也是细胞膜修复中的重要组成部分。在啮齿类动物和大多数动物模型中,MG53蛋白可以保护多种细胞膜免受破坏,同时也能改善肌营养不良、急性肺损伤、心肌梗死和急性肾脏损伤等疾病,而且静脉注射MG53蛋白能够透过血脑屏障,从而保护脑缺血性损伤。然而,目前有关重组人MG53蛋白联合hUC-MSCs治疗TBI的研究尚未见报道。此外,MG53蛋白能否单独对损伤的神经元细胞发挥保护作用也未见报道。目的本课题旨在探究MG53蛋白对神经元细胞的保护作用以及其联合hUC-MSCs对TBI小鼠的保护作用。建立脂多糖（Lipopolysaccharide,LPS）诱导的小鼠海马神经元HT22细胞损伤模型,研究MG53蛋白对HT22增殖、凋亡、周期、迁移、抗氧化的影响及其分子机制。以TBI小鼠为研究对象,探讨MG53蛋白联合hUC-MSCs移植对TBI小鼠的治疗作用和分子机制。方法第一部分研究MG53蛋白对LPS诱导的HT22细胞损伤的保护作用1、CCK-8法检测细胞增殖,筛选LPS作用于HT22细胞的最佳浓度及时间。实验分为正常对照组（Normal Control,NC组）、MG53组、LPS组和MG53+LPS组。2、采用流式细胞术检测MG53蛋白对HT22细胞周期与凋亡的影响。3、划痕实验检测MG53蛋白对HT22细胞迁移的影响;ELISA检测细胞SOD活力和MDA含量变化。4、qRT-PCR检测MG53蛋白对HT22细胞TNF-α、IL-6、IL-1β、TLR4、MyD88和NF-κB mRNA表达的影响。5、Western Blot检测凋亡相关蛋白Bax、Bcl-2、细胞周期蛋白（Cyclin D1）及TLR4/NF-κB信号通路相关蛋白等的表达变化。第二部分探讨MG53蛋白联合hUC-MSCs治疗TBI的作用及机制1、C57BL/6小鼠建立TBI模型,随机分为TBI组、MG53组、MSCs组和MG53+MSCs组。2、术后3天,PI染色检测损伤部位皮质区细胞死亡情况,干湿重法检测脑水肿情况。3、Western Blot检测小鼠病灶部位MG53蛋白、凋亡相关蛋白Bcl-2和Bax以及促生存通路相关蛋白p-Akt1,p-GSK3β和p-ERK1/2的表达变化。4、免疫荧光检测海马区MAB1281表达情况,ELISA试剂盒检测小鼠血清中SOD活力和MDA含量。5、术后1-28天,mNSS评估小鼠神经功能恢复情况、Morris水迷宫实验检测小鼠学习记忆能力;术后28天,新事物识别实验、糖水偏好实验和强迫游泳实验检测小鼠的抑郁程度。6、CV染色检测小鼠脑损伤体积,qRT-PCR检测BDNF、NGF的表达;术后14天、28天,免疫荧光检测海马区DCX、NeuN表达情况。结果第一部分MG53蛋白对LPS诱导的HT22细胞损伤的保护作用及其机制初步研究1、CCK8结果显示,LPS诱导对HT22细胞增殖具有浓度和时间依赖双向效应,即低浓度水平促进细胞增殖,高浓度水平抑制细胞增殖,引起细胞凋亡（P<0.05）,其中500mg/L LPS作用HT22细胞48 h后,细胞抑制率接近50%,用于后续实验模型的建立。2、与LPS组相比,MG53+LPS组细胞存活率升高,凋亡率降低,G₀/G₁期减少,S期增加,迁移能力增加（P<0.05）。Western Blot结果显示,与LPS组相比,MG53+LPS组细胞中Bcl-2蛋白表达升高,Bax蛋白表达降低,Cyclin D1表达升高（P<0.05）。3、ELISA结果显示,与NC组相比,LPS组细胞SOD活性降低,MDA含量升高,与LPS组相比,MG53+LPS组细胞的SOD活性升高,MDA含量降低,差异具有统计学意义（P<0.05）。4、Western Blot结果显示,与NC组相比,LPS组TNF-α,IL-6和IL-1β蛋白表达上调（P<0.05）,TLR4/NF-κB信号通路中总的NF-κB p65蛋白表达无显著变化（P>0.05）,IKBα蛋白表达水平降低（P<0.05）,TLR4,p-NF-κB p65,p-IKBα蛋白表达升高（P<0.05）;与LPS组相比,LPS+MG53组TNF-α,IL-6,IL-1β蛋白表达水平降低（P<0.05）,总的NF-κB p65表达无显著变化（P>0.05）,IKBα表达升高,TLR4,p-NF-κB p65,p-IKBα蛋白表达降低（P<0.05）,qRT-PCR的结果与Western Blot的结果一致（P<0.05）。第二部分MG53蛋白联合hUC-MSCs治疗小鼠创伤性脑损伤1、术后3天,与TBI组相比,各治疗组小鼠的大脑病灶侧含水量明显降低,联合治疗组小鼠脑含水量最低（P<0.05）,病灶对侧脑含水量无显著差异（P>0.05）;与TBI组相比,各治疗组小鼠的大脑皮质PI阳性细胞减少,抑凋亡蛋白Bcl-2表达升高,促凋亡蛋白Bax表达降低,而联合治疗组变化最明显（P<0.05）。2、免疫荧光结果显示,MSCs组和MG53+MSCs组能够检测到MAB1281阳性细胞,TBI组和MG53组未检测到MAB1281阳性细胞;与TBI组相比,各治疗组小鼠血清中自由基清除剂SOD活力升高,氧化应激产物MDA含量降低,且联合治疗组的效果更明显（P<0.05）。3、Western结果显示,MG53组和MG53+MSCs组能够检测到MG53蛋白,而TBI组和MSCs组未检测到MG53蛋白;与TBI组相比,MG53组p-Akt1和p-ERK1/2的表达显著升高（P<0.05）,p-GSK3β无显著变化（P>0.05）,MSCs组和MG53+MSCs组的p-Akt1,p-GSK3β和p-ERK1/2的表达均显著升高（P<0.05）,而且联合治疗组变化更明显（P<0.05）,但是总的Akt1,GSK3β和ERK1/2的表达没有显著变化（P>0.05）,提示MG53蛋白和hUC-MSCs可能是通过调控PI3K/Akt-GSK3β和ERK1/2信号通路对脑损伤小鼠起保护作用。4、术后各组小鼠体重在1天、3天减轻,从7天开始增加。术后7天后,与TBI组相比,各治疗组小鼠体重明显增加,mNSS评分明显降低,且联合治疗组小鼠体重增加最快,mNSS评分最低（P<0.05）;与TBI组相比,各治疗组小鼠穿越平台次数增加,平台所在象限停留时间增加,逃避潜伏期时间缩短,悬浮时间减少,辨别指数和糖水偏好度均显著提高（P<0.05）,且联合治疗组的效果更明显（P<0.05）。5、与TBI组相比,各治疗组小鼠神经营养因子BDNF、NGF mRNA表达升高（P<0.05）,且联合治疗组表达最高（P<0.05）;免疫荧光结果显示,与TBI组相比,各治疗组小鼠海马区DCX和NeuN阳性细胞增加（P<0.05）,联合治疗组阳性细胞数增加更显著（P<0.05）。结论1、MG53蛋白能够促使LPS损伤的HT22细胞存活,抑制细胞凋亡,促使细胞从G0/G1期向S期运转,提高细胞迁移力和抗氧化能力,抑制TLR4/NF-κB信号通路,降低LPS诱导的炎症反应。2、MG53蛋白和hUC-MSCs能够透过血脑屏障迁移到TBI小鼠损伤部位,治疗后能够增加小鼠体重,改善认知功能,增强学习记忆能力,提高神经恢复,减轻抑郁,抑制细胞凋亡,促进神经再生,提高抗氧化水平,而且联合治疗比单一治疗效果更加明显。此外,MG53蛋白和hUC-MSCs可能通过激活PI3K/Akt-GSK3β和ERK1/2信号通路对TBI小鼠发挥保护作用。更多还原

【Abstract】 Traumatic brain injury(TBI)is a common traumatic disease caused by external force,which causes brain nerve dysfunction and nerve cell death.It can also cause severe physical,psychological and cognitive impairments,therefore a new therapy for TBI is in critical need.The emergence of stem cells and regenerative medicine will provide new ideas for the treatment of TBI.Several experimental and clinical studies have shown that stem cell transplantation exerted beneficial effects in TBI,via paracrinefactors and cell replacement.Human umbilical cord derived mesenchymal stem cells(h UC-MSCs)have fast rate of self-renewal,low immunogenicity,and low risk of teratoma formation,supporting their therapeutic value.However,the migration and survival of transplanted stem cells are often hindered because of the pathology and cell death that accompanies TBI caused by excessivereactive oxygen species generation associated with tissue ischemia and inflammation.Mitsugumin53(MG53)protein is a member of the tripartite motif(TRIM)family proteins.It has an essential role in cell membrane repair and recovery.MG53 protein can protect various cell types against membrane disruption when applied to the extracellular environment and ameliorate the pathologies associated with muscular dystrophy,acute lung injury,myocardial infarction,and acute kidney injury in rodent and large animal models of these diseases.Additionally,intravenous delivery of MG53 was able to permeate through the blood-brain barrier to protect against ischemic brain injury.However,there is no report about the research of MG53 protein combined with h UC-MSCs in the treatment of TBI.In addition,the protective effect of MG53 protein on damaged neuron cells has not been reported.Objective In the present study we aim to explore the protective effects of MG53 protein on injured neuron cells in vitro and the protective effects of MG53 combined with h UC-MSCs transplantation in a mouse model of traumatic brain injury in vivo.Part I: To determine the HT22 cell damage model of hippocampal neurons induced by Lipopolysaccharide and to explore the effect of MG53 on HT22 cell proliferation,apoptosis,cycle,migration and its molecular mechanism.Part II: To investigate the protective effects of MG53 in combination with h UC-MSCs as well as its underlying biological mechanisms in vivo.Methods Part Ⅰ Protective effect of MG53 protein on HT22 cells damaged by LPS 1.CCK-8 assay was used to detect cell proliferation and determine the optimal concentration and time of LPS to induce HT22 cells injuried.The experiment was divided into normal control group(NC group),MG53 group,LPS group and MG53+LPS group.2.After LPS treatment,cell cycle was measured by flow cytometry and Annexin V-FITC/PI method to detect cell apoptosis.3.Scratching experiment was used to detect the migration distance of cells.ELISA kit was used to detect changes in cell SOD activity and MDA content.4.Quantitative real time PCR were used to detect the m RNA levels of the TNF-α,IL-6,IL-1β,TLR4,My D88 and NF-κB expression.5.Western Blot was used to detect the expression of Bax,Bcl-2,cyclin D1 and the activity of TLR4/NF-κB signaling pathway.Part Ⅱ MG53 protein combined with h UC-MSCs in the treatment of TBI and its molecular mechanism 1.C57BL/6 mice subjected to TBI were random Ly divided into 4 groups: TBI group,MG53 group,MSCs group and MG53+MSCs group.2.At day 3 post TBI,PI staining was used to detect the damage of cortical cell death;brain edema was detected by dry wet method.3.Western Blot was used to detect the expression of Bax,Bcl-2,MG53 and pro-survival pathway related protein p-Akt1,expression of p-GSK3β and p-ERK1/2.4.Immunofluorescence detection of hippocampal MAB1281 expression;SOD activity and MDA content in mice serum were tested by ELISA.5.Between 1 and 28 days,the m NSS score was used to evaluate the recovery of neurological function in mice;Between 21 and 28 days,the Morris water maze test to determine the learning and memory ability of mice;At day 28 post TBI,the new recognition test,sucrose preference test and forced swimming test to determine the degree of depression of mice.6.CV staining was used to detect the volume of brain injury in mice;RT-PCR were used to detect the m RNA levels of the BDNF and NGF expression;At days 14 and 28,mices were sacrificed and brain tissue was prepared to assess neurogenesis by immunofluorescent staining of DCX and Neu N,respectively.Results Part Ⅰ Protective effects of MG53 protein on HT22 cells damaged by LPS and its mechanism 1.LPS stimulation had a concentration and time dependent effect on the proliferation of HT22 cells.That was,low concentration level promoted cell proliferation,and high concentration inhibited cell proliferation and induced cell apoptosis(P<0.05).CCK-8 results showed that after 500mg/L LPS induced HT22 cells 48 h,the cell growth inhibition rate was close to 50%,which could be used for subsequent experimental models.2.Compared with LPS group,cell survival rate increased,apoptosis rate decreased,G0/G1 phase decreased,S phase increased and migration capacity increased(P<0.05)in MG53+LPS group.Western results showed that compared with LPS group,the expression of Bcl-2 protein expression increased,Bax protein expression decreased,Cyclin D1 expression increased in MG53+LPS group(P<0.05).3.ELISA results showed that compared with the NC group,the SOD activity decreased and the MDA content increased in LPS group.Compared with the LPS group,the SOD activity increased and the MDA content decreased in the MG53+LPS group,and the difference was statistically significant(P<0.05).4.Western Blot results showed that compared with the NC group,the TNF-α,IL-6,IL-1β,TLR4,p-NF-k Bp65,p-IKBα protein expression increased(P<0.05),the total NF-κBp65 protein expression was not significantly changed(P>0.05),and the IKBα protein expression decreased in LPS group(P<0.05).Compared with the LPS group,the TNF-α,IL-6,IL-1β,TLR4,p-NF-k Bp65,p-IKBα protein expression decreased(P<0.05),the total NF-κB p65 protein expression was not significantly changed(P>0.05),and the IKBα protein expression increased in LPS+MG53 group(P<0.05).The result of q RT-PCR is consistent with the result of Western Blot(P<0.05).Part Ⅱ MG53 protein combined with h UC-MSCs in treatment of TBI and its molecular mechanism 1.After operation of 3 days,compared with the TBI group,the brain water content of mice in treatment groups was significantly lower,and the MG53+MSCs group was the lowest(P<0.05),while there was no significant difference in the contralateral brain(P>0.05).Compared with the TBI group mices,the PI positive cells in the cerebral cortex of each treatment mice decreased,the expression of Bcl-2 protein increased,Bax protein decreased,and the change of the MG53+MSCs group was the most obvious(P<0.05).2.Immunofluorescence showed that MAB1281 positive cells could be detected in MSCs and MG53+MSCs groups,but no MAB1281 positive cells were found ingroup TBI and MG53 groups.Compared with the TBI group,the SOD activity of mice in each treatment groups increased,the MDA content of mice in each treatment groups decreased,and the effect of the MG53+MSCs group was better(P<0.05).3.Western Blot results showed that MG53 protein could be detected in MG53 and MG53+MSCs groups,while MG53 was not detected in TBI and MSCs groups.Compared with the TBI group,the expression of p-Akt1 and p-ERK1/2 increased significantly in MG53 group(P<0.05),and there was no significant change of p-GSK3β in MG53 group(P>0.05).The expressions of p-Akt1,p-GSK3β and p-ERK1/2 were significantly increased in MSCs and MG53+MSCs groups,and the changes in the MG53+MSCs group were more obvious.It is suggested that MG53 and h UC-MSCs may play an essential role in brain damage mice by regulating PI3K/Akt-GSK3β and ERK1/2 signaling pathways.4.After operation of day 1 and 3,the weight of mice in each group decreased,and the weight began to increase from day 7.After 7 days,compared with the TBI group,the body weight of mice in each treatment group increased,the m NSS score decreased,and the MG53+MSCs group was more significant(P<0.05).Compared with the TBI group,the number of cross platform in each treatment group increased,the resting time of the platform increased,the escape latency shortened,the suspension time decreased,the discrimination index and the sugar water preference increased significantly(P<0.05),while the effect of the MG53+MSCs group was better(P< 0.05).5.Compared with the TBI group,the expression of neurotrophic factor BDNF and NGF m RNA in each treatment groups increased(P<0.05),while the effect of MG53+MSCs group was better(P<0.05).The results of immunofluorescence showed that compared with the TBI group,the positive cells of DCX and Neu N in the hippocampus of each treatment groups increased(P<0.05),while the DCX and Neu N in the MG53+MSCs group were more significant(P<0.05).Conclusion 1.MG53 protein can promote the survival of HT22 cells damaged by LPS,inhibit cell apoptosis,promote cell operation from G0/G1 to S phase,improve cell migration and antioxidant capacity,inhibit TLR4/NF-k B signaling pathway,and reduce LPS induced inflammatory response.2.MG53 protein and h UC-MSCs can migrate through the blood-brain barrier to the injured part of TBI mices,increase the weight of the mices,improve the cognitive function,enhance the learning and memory ability,improve the nerve recovery,reduce the depression,inhibit the apoptosis,promote the nerve regeneration and antioxidant level,while the MG53+MSCs treatment is more obvious than the single treatment.In addition,MG53 protein and h UC-MSCs may play a protective role in TBI mices by activating PI3K/Akt-GSK3β and ERK1/2 signaling pathways.更多还原