自体血管内皮祖细胞抗衰老作用的初步临床研究

发布时间：2018-02-28 16:26

本文关键词： 自体血管内皮祖细胞超氧化物歧化酶胰岛素生长因子-1 抗衰老老龄化　出处：《南方医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：[背景] 老龄化问题被认为是21世纪三大世界性社会问题之一。WHO对老龄化的定义是如果一个国家的60岁以上老年人口占人口总数的10%,或65岁以上老年人口占总人口数的7%,即这个国家已经步入老龄化阶段。发达国家是最早步入老龄化阶段的国家,自20世纪70年代以来,世界各国人口年龄结构的老龄化过程存在着差异。截至2006年底,我国已经是老龄化人口唯一超过1亿的国家,到2050年我国衰老人口将占总人口的四分之一。目前研究认为衰老和老年病的发生是相关联的,二者具有相同的病理生理过程,如免疫系统损伤、神经内分泌紊乱、脂类及糖类代谢紊乱等。衰老后一些老年病如动脉粥样硬化、冠心病、糖尿病、高血压病、神经变性疾病等的发生率明显增加。因此衰老将导致老年病的发生率增加、患病率提高。这些老年病将严重影响老年人群的健康,需要占用更多的医疗资源,给我国经济及生产力带来极大的冲击。因此延缓衰老对我国目前的发展具有重要意义。有关衰老机制的研究很多,目前已经深入到分子水平。如p16-INK4a被认为是一种细胞衰老的重要生物学标记,清除p16-INK4a阳性的细胞可以延缓衰老。细胞衰老涉及到多种复杂的生物分子机制,如能实现再生细胞代替衰老细胞,使细胞的再生和衰老实现均衡,延缓衰老有可能成为现实。1493-1541年,菲律宾Auredus Paracelsus首次提出细胞疗法,通过输入活细胞使机体获得年轻化,自20世纪50年代,全世界已经有数百万人接受细胞疗法。细胞疗法其中之一即是干细胞疗法。衰老进程中干细胞不断丢失,因此急时补充干细胞有可能延缓衰老。目前脂肪干细胞抗衰老已经初步应用于临床并取得确切疗效,骨髓间充干细胞等在动物实验组已被证实有抗衰老作用,目前血管内皮祖细胞(Endothelial progenitor cells, EPCs)已经在急性心肌梗塞、难治性心绞痛、肢体缺血、慢性肝病等缺血性疾病的临床实验得到应用。Taguichi的临床试验证实血管内皮祖细胞不仅可以促进脑缺血部位的血管新生,维持血管内皮细胞的功能,还可以促进神经元的生长。以上组织和功能的恢复与血管内皮祖细胞分泌的内皮生长因子、胰岛素生长因子及纤维母细胞生长因子等有关。据研究报道胰岛素生长因子具有抗衰老的作用,而血管内皮祖细胞是否具有抗衰老作用,目前临床研究中尚未见到报道。目前应用于抗衰老评定的主要有抗氧化类物质、激素类、免疫功能及量表。自由基衰老理论认为细胞代谢产生的自由基,在人体衰老中起很重要的作用。超氧化物歧化酶(Superoxide dismutase, SOD)是体内自由基防御体系中最重要的酶类抗氧化剂,它可直接清除超氧阴离子自由基,阻断自由基连锁反应,保护组织免受超氧阴离子自由基的损伤,SOD活力的高低间接反应机体抗氧化的能力,即反应机体抗衰老能力；人体生长、发育与衰老主要受激素分泌水平的调节,激素分泌水平在不同的生理阶段发生不同的变化,IFG-1具有抗衰老作用,能反应机体衰老程度；衰老的免疫学假说认为免疫系统参加了脊椎动物的衰老过程,是衰老过程的主要调节系统之一,可以通过免疫球蛋白(IgA、IgG、IgM)测定免疫系统机能,随着衰老,血液中IgA、IgG升高,IgM降低；量表如衰老自测量表及匹兹堡睡眠量表评定可以测定机体机能状态；生理耗能指数(physiological index of energy consumption, PCI)亦能评定机体机能状态, [目的] 采用血液指表及量表评定观察输入自体血管内皮祖细胞的安全性及其在抗衰老方面的作用。 [方法] 2011-10至2012-10月在我院康复科选取4名受试者。入选患者：25岁年龄80岁；自愿配合治疗及随访；排除标准：孕妇、哺乳期患者及肿瘤病患,自身免疫性疾病,对青霉素等过敏,发热性疾病,肝肾呼吸系统疾病者。实验组2例(男1例45岁,女1例75岁),对照组2例(男1例49岁,女1例77岁)。实验组和对照组均行常规治疗(传统中医治疗及运动疗法),此外实验组进行自体EPCs移植。治疗方法： 1.实验前实验组和对照组均进行血常规、血凝指标、尿常规、便常规、肝肾功、糖代谢指标、脂代谢指标、心衰指标、炎症指标、传染病八项定量相关检查,排除不能纳入临床研究的对象。 2.血管内皮祖细胞获取实验组行骨髓穿刺：均选用髂前上棘抽取骨髓50m1左右,行干细胞培养。自体肱静脉外周血30m1,离心获得血清后冰冻保存以备混悬干细胞用。对照组无需行此操作。 3.细胞培养及鉴定细胞培养严格在特定条件下进行,密度梯度离心法获取骨髓单个核细胞。将骨髓单个核细胞以1×105/cm2接种于纤连蛋白包被的塑料培养瓶。特定培养液进行培养,培养4天后去除非粘附细胞。每周换液2次,细胞80%融合时进行传代。一般血液培养需一月左右可达到移植需求量(5×106/kg),细胞培养后浓度及分离均由公司达到统一标准。专业公司进行端粒酶表达情况、表面标志、成品检定、存活率、内毒素、无菌检查、支原体、病毒外源因子、逆转录病毒、特殊人源病毒、细胞致瘤性、残余血清检测等。 4.细胞移植细胞扩增到足够的数量后(5×106/kg),进行移植。第一次移植,将2.5×106/kg个细胞,混悬于含5%自体血清的生理盐水100ml中,经桡静脉输入体内。输入过程中密切观察患者反应,如有不良反应时,急时停止输液。约1周后行第二次移植,移植的细胞数量同第一次。 5.实验前及试验后第4个月采集受试者血液指标SOD、IFG-1、免疫球蛋白三项IgA、IgG、IgM,量表评定受试者衰老程度及睡眠质量,测定机体生理耗能指数。 [疗效评定标准] 1.超氧化物歧化酶(SOD) 2.胰岛素样生长因子-1(IGF-1) 3.免疫球蛋白三项IgA、IgG、IgM 4.衰老自测量表(AAS)量表评定 5.匹兹堡睡眠指数(PSQI) 6.生理耗能指数(PCI) [统计学方法] 暂无：本实验目前纳入受试对象共4例。预期采用SPSS13.0统计软件对实验结果进行统计学处理。 [结果] 1.超氧化物歧化酶(SOD) 实验组两名受试者治疗后血液中超氧化物歧化酶歧化酶浓度较治疗前升高,对照组受试者治疗后较治疗前血液中SOD,Ⅲ降低,Ⅳ升高。 2.胰岛素样生长因子-1(IGF-1) 实验组两名受试者治疗后血液中胰岛素样生长因子-1(IGF-1)较治疗前升高,对照组两名受试者治疗后血液中胰岛素样生长因子-1(IGF-1)较治疗前变化不明显。 3.免疫球蛋白IgA、IgG、IgM 实验组两名受试者治疗后较治疗前血液中IgA、IgG降低,IgM升高,对照组两名受试者血液治疗后较治疗前血液中IgA、IgG下降,IgM无明显变化。 4.衰老自测量表(AAS)量表评定实验组两名受试者治疗后较治疗前量表评定结果有明显降低；对照组两名患者治疗后较治疗前量表评定有降低,变化量较实验组小。 5.匹兹堡睡眠量表(PSQI)量表实验组两名受试者治疗后较治疗前量表评定结果降低；对照组两名患者治疗后较治疗前量表评定降低。 6.生理耗能指数(PCI) 实验组两名受试者治疗后较治疗前量表评定结果有明显降低；对照组两名患者治疗后较治疗前量表评定有降低,变化量较实验组小。 [结论] 通过临床观察,输自体血管内皮祖细胞者的免疫系统功能,抗氧化能力,激素分泌水平,生理耗能指数及衰老自测水平等有改善。自体血管内皮祖细胞在治疗中安全可靠,治疗过程中及随访中未见免疫排斥反应及其它不良反应。自体血管内皮祖细胞将有可能成为抗衰老的一项新的研究课题。
[Abstract]:[background]
The problem of aging is considered to be one of the twenty-first Century three world social issues of.WHO on aging is defined as if a country's elderly population over the age of 60 accounted for 10% of the total population, or 65 years old population accounted for 7% of the total population, the country has entered the stage of aging in developed countries is the earliest. Entered the aging stage, since 1970s, differences in the aging process of population age structure of all countries in the world. As of the end of 2006, China has an ageing population is only more than 100 million countries, to 2050 the aging population in China accounted for 1/4 of the total population.
The present study of aging and age-related diseases are related, the two have the same pathological process, such as the immune system injury, neuro endocrine disorders, lipid and carbohydrate metabolism disorder after aging. Some senile diseases such as atherosclerosis, coronary heart disease, diabetes, hypertension, the incidence of neurodegenerative diseases increased significantly. The aging in the elderly disease incidence increased, the prevalence rate of senile disease increased. These will seriously affect the health of the elderly people, need more medical resources, has great impact on China's economy and productivity. So the aging is very important for the development of our country at present.
A lot of research about the mechanism of aging, has been to the molecular level. P16-INK4a is considered to be an important biological marker for cell senescence, removal of p16-INK4a positive cells can delay senescence. Cell senescence involves many complicated mechanisms of biological molecules, such as can realize the regeneration of cells to replace the aging cells, cell regeneration and aging to achieve a balanced, aging may become a reality in.1493-1541, Philippines Auredus Paracelsus first proposed by input cell therapy, living cells make the body get younger, since 1950s, millions of people around the world have already received cell therapy. Cell therapy, one of which is stem cell therapy. Stem cells being lost in aging process, so anxious supplementary stem cells have the potential to delay senescence. The fat stem cell aging has been applied to the clinic and proved to be effective, Bone marrow mesenchymal stem cells in animal experimental group have been confirmed to have anti-aging effect, the vascular endothelial progenitor cells (Endothelial progenitor cells, EPCs) in acute myocardial infarction, refractory angina, limb ischemia, clinical trials of chronic liver disease such as ischemic disease by vascular endothelial progenitor cells can promote cerebral ischemic areas angiogenesis is confirmed in clinical trials using.Taguichi, maintaining the function of vascular endothelial cells, but also can promote the growth of neurons. The organization and function of endothelial secretion restoration and endothelial progenitor cell growth factor, insulin-like growth factor and fibroblast growth factor. According to the research reports of insulin-like growth factor has anti-aging effect however, endothelial progenitor cells have anti-aging effect, is not seen currently in clinical research reports.
Antioxidant substances, mainly used in the evaluation of anti-aging hormones, immune function and scale. The free radical theory of aging cell metabolism of free radicals, plays a very important role in human aging. Superoxide dismutase (Superoxide dismutase SOD) is the most important in free radical defense system the antioxidant enzymes, it can scavenge superoxide anion free radical, blocking free radical chain reaction, protect the tissue from superoxide anion free radical damage, antioxidant ability of indirect reaction level of SOD activity, which should be anti anti-aging ability; human growth, development and senescence is mainly regulated by hormone, hormone secretion different changes in different physiological stages, IFG-1 has anti-aging effect, reaction of aging degree; immunology hypothesis of aging that immune system in The aging process of vertebrates, is one of the main control system of the aging process, the immunoglobulin (IgA, IgG, IgM) determination of immune system function, with the aging, blood IgA, IgG increased, IgM decreased; scale such as aging self testing scale and scale Pittsburgh sleep can determine the body function; physiological cost index (physiological index of energy consumption, PCI) can also assess their body function,
[Objective]
The safety of autologous vascular endothelial progenitor cells and its role in antiaging were evaluated by blood index and scale.
[method]
2011-10 to 2012-10 months in our hospital rehabilitation department selected 4 subjects. Patients: 25 years old 80 years of age; voluntary coordination of treatment and follow-up; exclusion criteria: pregnant, lactating patients and cancer, autoimmune diseases, allergic to penicillin, febrile diseases, and respiratory diseases. 2 cases in the experimental group (1 cases of male female 1 cases aged 45, 75 years old), 2 cases in the control group (1 cases of male 49, female 1 cases aged 77). The experimental group and the control group were treated with conventional therapy (traditional Chinese medicine therapy and exercise therapy), in addition to the experimental group and autologous EPCs transplantation.
Treatment methods:
1. before the experiment, both the experimental group and the control group underwent routine blood tests, blood coagulation indicators, urine routine, routine tests, liver and kidney function, glycometabolism index, lipid metabolism index, heart failure index, inflammatory index and infectious disease eight quantitative correlation tests, excluding those who could not be included in clinical research.
2. endothelial progenitor cells were obtained from the experimental group. Bone marrow puncture was performed. All the anterior superior iliac spine were selected for bone marrow stem cell culture. 50m1 and 30m1 were collected from the peripheral blood of the brachial vein. After centrifugation, the serum was cryopreserved for suspending stem cells. The control group did not need to perform this operation.
3. cell culture and identification of cell culture strictly under specific conditions, the method of density gradient centrifugation of bone marrow mononuclear cells. Bone marrow mononuclear cells with 1 x 105/cm2 seeded on fibronectin coated plastic bottle. Specific medium, cultured for 4 days to remove non adhesive liquid cells. Every week for 2 time for 80% cell fusion. General blood culture can reach the demand for transplant January around (5 * 106/kg), cell culture after concentration and separation by the company to achieve uniform standards. Professional company telomerase expression, surface markers, product verification, survival rate, endotoxin, sterility test, mycoplasma and exogenous virus factor, retrovirus, special human virus, cell tumorigenicity, residual serum detection.
4. cell transplantation cells amplified enough quantity (5 * 106/kg), for transplantation. The first transplantation, 2.5 * 106/kg cells, mixed saline with 5% 100ml suspension of autologous serum in the radial vein in vivo. Input the input process and closely observe the patient responses, such as adverse reaction, stop emergency infusion after the second transplantation. About 1 weeks, the number of cell transplantation at the first time.
5. blood samples were collected before and fourth months after the experiment. Subjects' blood index SOD, IFG-1, immunoglobulin three IgA, IgG, IgM were used to assess the senility and sleep quality of the subjects, and the physiological energy consumption index of the subjects was measured.
[therapeutic evaluation standard]
1. superoxide dismutase (SOD)
2. insulin like growth factor -1 (IGF-1)
3. immunoglobulin three IgA, IgG, IgM
Evaluation of the 4. senescence scale (AAS) scale
5. Pittsburgh sleep index (PSQI)
6. physiological energy consumption index (PCI)
[statistical method]
No: there are 4 subjects in this experiment. We expect to use SPSS13.0 statistical software to deal with the results of the experiment.
[results]
1. superoxide dismutase (SOD)
The concentration of superoxide dismutase dismutase in blood of the two subjects in the experimental group increased compared with that before treatment. After treatment, the levels of SOD and III in the control group decreased, while those in the control group increased.
2. insulin like growth factor -1 (IGF-1)
The insulin like growth factor -1 (IGF-1) in the experimental group increased after treatment, and the insulin like growth factor -1 (IGF-1) in the blood of the two subjects in the control group did not change significantly after treatment compared with that before treatment. The plasma level of insulin like growth factor -1 (IGF-1) in the control group was not significantly changed after treatment.
3. immunoglobulin IgA, IgG, IgM
After treatment, two subjects in the experimental group decreased blood IgA, IgG and IgM after treatment, while two subjects in the control group decreased blood IgA and IgG compared with that before treatment, but IgM did not change significantly.
Evaluation of the 4. senescence scale (AAS) scale
In the experimental group, two subjects were significantly lower than those before treatment, while two patients in the control group were lower than those before treatment.
5. Pittsburgh Sleep Scale (PSQI scale)
After treatment, the results of two subjects in the experimental group were lower than those before the treatment. Two patients in the control group were lower than the pre treatment scale after treatment.
6. physiological energy consumption index (PCI)
In the experimental group, two subjects were significantly lower than those before treatment, while two patients in the control group were lower than those before treatment.
[Conclusion]
Through clinical observation, antioxidant function, immune system loses the autologous endothelial progenitor cells, hormone secretion, physiological index and energy consumption level is improved. The aging self testing of autologous endothelial progenitor cells in the treatment of safety, during the treatment and follow-up no immune rejection and other adverse reactions of autologous vascular endothelial. Progenitor cells will likely become a new research topic in anti-aging.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329

【参考文献】