本文關(guān)鍵詞： 自體血管內(nèi)皮祖細(xì)胞 超氧化物歧化酶 胰島素生長(zhǎng)因子-1 抗衰老 老齡化　出處：《南方醫(yī)科大學(xué)》2013年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：[背景] 老齡化問(wèn)題被認(rèn)為是21世紀(jì)三大世界性社會(huì)問(wèn)題之一。WHO對(duì)老齡化的定義是如果一個(gè)國(guó)家的60歲以上老年人口占人口總數(shù)的10%,或65歲以上老年人口占總?cè)丝跀?shù)的7%,即這個(gè)國(guó)家已經(jīng)步入老齡化階段。發(fā)達(dá)國(guó)家是最早步入老齡化階段的國(guó)家,自20世紀(jì)70年代以來(lái),世界各國(guó)人口年齡結(jié)構(gòu)的老齡化過(guò)程存在著差異。截至2006年底,我國(guó)已經(jīng)是老齡化人口唯一超過(guò)1億的國(guó)家,到2050年我國(guó)衰老人口將占總?cè)丝诘乃姆种弧?目前研究認(rèn)為衰老和老年病的發(fā)生是相關(guān)聯(lián)的,二者具有相同的病理生理過(guò)程,如免疫系統(tǒng)損傷、神經(jīng)內(nèi)分泌紊亂、脂類(lèi)及糖類(lèi)代謝紊亂等。衰老后一些老年病如動(dòng)脈粥樣硬化、冠心病、糖尿病、高血壓病、神經(jīng)變性疾病等的發(fā)生率明顯增加。因此衰老將導(dǎo)致老年病的發(fā)生率增加、患病率提高。這些老年病將嚴(yán)重影響老年人群的健康,需要占用更多的醫(yī)療資源,給我國(guó)經(jīng)濟(jì)及生產(chǎn)力帶來(lái)極大的沖擊。因此延緩衰老對(duì)我國(guó)目前的發(fā)展具有重要意義。 有關(guān)衰老機(jī)制的研究很多,目前已經(jīng)深入到分子水平。如p16-INK4a被認(rèn)為是一種細(xì)胞衰老的重要生物學(xué)標(biāo)記,清除p16-INK4a陽(yáng)性的細(xì)胞可以延緩衰老。細(xì)胞衰老涉及到多種復(fù)雜的生物分子機(jī)制,如能實(shí)現(xiàn)再生細(xì)胞代替衰老細(xì)胞,使細(xì)胞的再生和衰老實(shí)現(xiàn)均衡,延緩衰老有可能成為現(xiàn)實(shí)。1493-1541年,菲律賓Auredus Paracelsus首次提出細(xì)胞療法,通過(guò)輸入活細(xì)胞使機(jī)體獲得年輕化,自20世紀(jì)50年代,全世界已經(jīng)有數(shù)百萬(wàn)人接受細(xì)胞療法。細(xì)胞療法其中之一即是干細(xì)胞療法。衰老進(jìn)程中干細(xì)胞不斷丟失,因此急時(shí)補(bǔ)充干細(xì)胞有可能延緩衰老。目前脂肪干細(xì)胞抗衰老已經(jīng)初步應(yīng)用于臨床并取得確切療效,骨髓間充干細(xì)胞等在動(dòng)物實(shí)驗(yàn)組已被證實(shí)有抗衰老作用,目前血管內(nèi)皮祖細(xì)胞(Endothelial progenitor cells, EPCs)已經(jīng)在急性心肌梗塞、難治性心絞痛、肢體缺血、慢性肝病等缺血性疾病的臨床實(shí)驗(yàn)得到應(yīng)用。Taguichi的臨床試驗(yàn)證實(shí)血管內(nèi)皮祖細(xì)胞不僅可以促進(jìn)腦缺血部位的血管新生,維持血管內(nèi)皮細(xì)胞的功能,還可以促進(jìn)神經(jīng)元的生長(zhǎng)。以上組織和功能的恢復(fù)與血管內(nèi)皮祖細(xì)胞分泌的內(nèi)皮生長(zhǎng)因子、胰島素生長(zhǎng)因子及纖維母細(xì)胞生長(zhǎng)因子等有關(guān)。據(jù)研究報(bào)道胰島素生長(zhǎng)因子具有抗衰老的作用,而血管內(nèi)皮祖細(xì)胞是否具有抗衰老作用,目前臨床研究中尚未見(jiàn)到報(bào)道。 目前應(yīng)用于抗衰老評(píng)定的主要有抗氧化類(lèi)物質(zhì)、激素類(lèi)、免疫功能及量表。自由基衰老理論認(rèn)為細(xì)胞代謝產(chǎn)生的自由基,在人體衰老中起很重要的作用。超氧化物歧化酶(Superoxide dismutase, SOD)是體內(nèi)自由基防御體系中最重要的酶類(lèi)抗氧化劑,它可直接清除超氧陰離子自由基,阻斷自由基連鎖反應(yīng),保護(hù)組織免受超氧陰離子自由基的損傷,SOD活力的高低間接反應(yīng)機(jī)體抗氧化的能力,即反應(yīng)機(jī)體抗衰老能力；人體生長(zhǎng)、發(fā)育與衰老主要受激素分泌水平的調(diào)節(jié),激素分泌水平在不同的生理階段發(fā)生不同的變化,IFG-1具有抗衰老作用,能反應(yīng)機(jī)體衰老程度；衰老的免疫學(xué)假說(shuō)認(rèn)為免疫系統(tǒng)參加了脊椎動(dòng)物的衰老過(guò)程,是衰老過(guò)程的主要調(diào)節(jié)系統(tǒng)之一,可以通過(guò)免疫球蛋白(IgA、IgG、IgM)測(cè)定免疫系統(tǒng)機(jī)能,隨著衰老,血液中IgA、IgG升高,IgM降低；量表如衰老自測(cè)量表及匹茲堡睡眠量表評(píng)定可以測(cè)定機(jī)體機(jī)能狀態(tài)；生理耗能指數(shù)(physiological index of energy consumption, PCI)亦能評(píng)定機(jī)體機(jī)能狀態(tài), [目的] 采用血液指表及量表評(píng)定觀察輸入自體血管內(nèi)皮祖細(xì)胞的安全性及其在抗衰老方面的作用。 [方法] 2011-10至2012-10月在我院康復(fù)科選取4名受試者。入選患者：25歲年齡80歲；自愿配合治療及隨訪；排除標(biāo)準(zhǔn)：孕婦、哺乳期患者及腫瘤病患,自身免疫性疾病,對(duì)青霉素等過(guò)敏,發(fā)熱性疾病,肝腎呼吸系統(tǒng)疾病者。實(shí)驗(yàn)組2例(男1例45歲,女1例75歲),對(duì)照組2例(男1例49歲,女1例77歲)。實(shí)驗(yàn)組和對(duì)照組均行常規(guī)治療(傳統(tǒng)中醫(yī)治療及運(yùn)動(dòng)療法),此外實(shí)驗(yàn)組進(jìn)行自體EPCs移植。 治療方法： 1.實(shí)驗(yàn)前實(shí)驗(yàn)組和對(duì)照組均進(jìn)行血常規(guī)、血凝指標(biāo)、尿常規(guī)、便常規(guī)、肝腎功、糖代謝指標(biāo)、脂代謝指標(biāo)、心衰指標(biāo)、炎癥指標(biāo)、傳染病八項(xiàng)定量相關(guān)檢查,排除不能納入臨床研究的對(duì)象。 2.血管內(nèi)皮祖細(xì)胞獲取實(shí)驗(yàn)組行骨髓穿刺：均選用髂前上棘抽取骨髓50m1左右,行干細(xì)胞培養(yǎng)。自體肱靜脈外周血30m1,離心獲得血清后冰凍保存以備混懸干細(xì)胞用。對(duì)照組無(wú)需行此操作。 3.細(xì)胞培養(yǎng)及鑒定細(xì)胞培養(yǎng)嚴(yán)格在特定條件下進(jìn)行,密度梯度離心法獲取骨髓單個(gè)核細(xì)胞。將骨髓單個(gè)核細(xì)胞以1×105/cm2接種于纖連蛋白包被的塑料培養(yǎng)瓶。特定培養(yǎng)液進(jìn)行培養(yǎng),培養(yǎng)4天后去除非粘附細(xì)胞。每周換液2次,細(xì)胞80%融合時(shí)進(jìn)行傳代。一般血液培養(yǎng)需一月左右可達(dá)到移植需求量(5×106/kg),細(xì)胞培養(yǎng)后濃度及分離均由公司達(dá)到統(tǒng)一標(biāo)準(zhǔn)。專(zhuān)業(yè)公司進(jìn)行端粒酶表達(dá)情況、表面標(biāo)志、成品檢定、存活率、內(nèi)毒素、無(wú)菌檢查、支原體、病毒外源因子、逆轉(zhuǎn)錄病毒、特殊人源病毒、細(xì)胞致瘤性、殘余血清檢測(cè)等。 4.細(xì)胞移植細(xì)胞擴(kuò)增到足夠的數(shù)量后(5×106/kg),進(jìn)行移植。第一次移植,將2.5×106/kg個(gè)細(xì)胞,混懸于含5%自體血清的生理鹽水100ml中,經(jīng)橈靜脈輸入體內(nèi)。輸入過(guò)程中密切觀察患者反應(yīng),如有不良反應(yīng)時(shí),急時(shí)停止輸液。約1周后行第二次移植,移植的細(xì)胞數(shù)量同第一次。 5.實(shí)驗(yàn)前及試驗(yàn)后第4個(gè)月采集受試者血液指標(biāo)SOD、IFG-1、免疫球蛋白三項(xiàng)IgA、IgG、IgM,量表評(píng)定受試者衰老程度及睡眠質(zhì)量,測(cè)定機(jī)體生理耗能指數(shù)。 [療效評(píng)定標(biāo)準(zhǔn)] 1.超氧化物歧化酶(SOD) 2.胰島素樣生長(zhǎng)因子-1(IGF-1) 3.免疫球蛋白三項(xiàng)IgA、IgG、IgM 4.衰老自測(cè)量表(AAS)量表評(píng)定 5.匹茲堡睡眠指數(shù)(PSQI) 6.生理耗能指數(shù)(PCI) [統(tǒng)計(jì)學(xué)方法] 暫無(wú)：本實(shí)驗(yàn)?zāi)壳凹{入受試對(duì)象共4例。預(yù)期采用SPSS13.0統(tǒng)計(jì)軟件對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)處理。 [結(jié)果] 1.超氧化物歧化酶(SOD) 實(shí)驗(yàn)組兩名受試者治療后血液中超氧化物歧化酶歧化酶濃度較治療前升高,對(duì)照組受試者治療后較治療前血液中SOD,Ⅲ降低,Ⅳ升高。 2.胰島素樣生長(zhǎng)因子-1(IGF-1) 實(shí)驗(yàn)組兩名受試者治療后血液中胰島素樣生長(zhǎng)因子-1(IGF-1)較治療前升高,對(duì)照組兩名受試者治療后血液中胰島素樣生長(zhǎng)因子-1(IGF-1)較治療前變化不明顯。 3.免疫球蛋白IgA、IgG、IgM 實(shí)驗(yàn)組兩名受試者治療后較治療前血液中IgA、IgG降低,IgM升高,對(duì)照組兩名受試者血液治療后較治療前血液中IgA、IgG下降,IgM無(wú)明顯變化。 4.衰老自測(cè)量表(AAS)量表評(píng)定 實(shí)驗(yàn)組兩名受試者治療后較治療前量表評(píng)定結(jié)果有明顯降低；對(duì)照組兩名患者治療后較治療前量表評(píng)定有降低,變化量較實(shí)驗(yàn)組小。 5.匹茲堡睡眠量表(PSQI)量表 實(shí)驗(yàn)組兩名受試者治療后較治療前量表評(píng)定結(jié)果降低；對(duì)照組兩名患者治療后較治療前量表評(píng)定降低。 6.生理耗能指數(shù)(PCI) 實(shí)驗(yàn)組兩名受試者治療后較治療前量表評(píng)定結(jié)果有明顯降低；對(duì)照組兩名患者治療后較治療前量表評(píng)定有降低,變化量較實(shí)驗(yàn)組小。 [結(jié)論] 通過(guò)臨床觀察,輸自體血管內(nèi)皮祖細(xì)胞者的免疫系統(tǒng)功能,抗氧化能力,激素分泌水平,生理耗能指數(shù)及衰老自測(cè)水平等有改善。自體血管內(nèi)皮祖細(xì)胞在治療中安全可靠,治療過(guò)程中及隨訪中未見(jiàn)免疫排斥反應(yīng)及其它不良反應(yīng)。自體血管內(nèi)皮祖細(xì)胞將有可能成為抗衰老的一項(xiàng)新的研究課題。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 馮藝萍;葛為公;;抗衰老藥物的使用現(xiàn)狀及研究進(jìn)展[J];華夏醫(yī)學(xué);2006年02期

2 李春明;劉毅;;脂肪干細(xì)胞及其在脂肪組織工程中的應(yīng)用[J];中國(guó)美容醫(yī)學(xué);2006年04期

3 韓曉琳,張宗玉,童坦君;衰老過(guò)程中原癌基因及抑癌基因的表達(dá)譜[J];生理科學(xué)進(jìn)展;2002年02期

4 李昕;張潔;宋佳霖;洪文學(xué);;傳統(tǒng)灸療法抗衰老研究進(jìn)展[J];生物醫(yī)學(xué)工程學(xué)雜志;2006年02期

5 劉孟章,李慶昌,衣紫桐,韓彩莉;生殖干細(xì)胞與衰老關(guān)系的研究(一)[J];現(xiàn)代中西醫(yī)結(jié)合雜志;2002年13期

6 羅利民;布林;;人臍血間質(zhì)干細(xì)胞靜脈輸注對(duì)~(60)Coγ射線照射小鼠脾臟細(xì)胞生物學(xué)效應(yīng)的影響[J];中國(guó)組織工程研究與臨床康復(fù);2009年01期

7 胡寒春;鄧云龍;范華;聶華林;;中老年人衰老自評(píng)問(wèn)卷的初步編制[J];中國(guó)臨床心理學(xué)雜志;2010年01期

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