移植脂肪源干细胞对D-半乳糖衰老大鼠的影响

发布时间：2018-01-12 21:34

本文关键词：移植脂肪源干细胞对D-半乳糖衰老大鼠的影响　出处：《南方医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 衰老是整个机体形态结构和生理机能的全面衰退,是一个动态的、复杂的过程。当前预防和延缓衰老已成为生命科学研究的热点。原林教授提出的“筋膜学假说”认为人体是由尚未分化的非特异性结缔组织(筋膜)支架所构成的支持与储备系统和以已分化功能细胞为基础的功能系统所共同构成。功能系统在支持与储备系统支持下维持结构与功能的稳定,支持与储备系统不断的为功能系统的细胞更新和功能活动提供源源不断的细胞源泉和细胞活动所需要的营养物质。功能系统是建立在功能细胞的专能特化基础上的,而支持与储备系统的筋膜支架是以干细胞为核心,通过分化出各种定向干细胞,为功能系统的更新提供源源不断的细胞补充,并为功能系统的各种细胞的更新、代谢提供一个稳定的内部环境。衰老是筋膜中干细胞储备逐渐耗竭的过程,而如何保持筋膜的正常状态,为功能系统提供稳定的细胞来源并维持其向功能细胞的正常分化,是延长生命周期的关键。如果衰老时减少的干细胞能得到及时的补充,为机体提供新的细胞储备,可修复机体损伤,延缓衰老、延长生命周期。间充质干细胞(menchymal stem cells)具有自我更新、多向分化的能力。最初是由Friendenstein等发现,且证明其在体外可以分化成为成骨细胞及脂肪细胞,而后其它的研究小组发现骨髓来源的MSCs不仅可以分化为中胚层来源的组织,而且可以分化为内胚层和外胚层来源的组织。间充质干细胞的多组织来源与筋膜结缔组织支架的全身分布是一致的,其纵向、横向分化的生物学特性与筋膜学提出的筋膜结缔组织对机体的支持储备作用也是一致的。ADSCs是筋膜中未分化干细胞的一种储备形式。ADSCs具有多分化潜能,能够跨胚层分化。ADSCs具有低免疫原性,免疫调节功能和分泌细胞因子等能力为异体移植提供了有利条件,使ADSCs成为生物治疗的亮点之一；ADSCs分泌细胞因子抗老化的作用,已经被临床广泛应用。ADSCs不仅可以用来进行各种退行性和衰竭疑难病的替代治疗,而且可用来进行辅助的免疫治疗。ADSCs可能通过以下机制修复或者重建组织：首先,ADSCs被植入一个受损的或是坏死的组织,它们能以副分泌的方式分泌出细胞活素和生长因子以刺激修复。ADSCs可以提供抗氧化剂、自由基清除剂、热休克蛋白等给待修复组织,从而清除局部环境中所释放的有毒物质,促进仍存活的细胞的修复。另外,ADSCs可以通过刺激内生性干细胞补充到待修复的脏器或自身直接迁移补充到待修复区域,以调节干细胞的微环境,并且促进它们沿着需要的方式分化为功能细胞,从而更新机体衰老的功能细胞,维持机体平衡。 D-gal所致亚急性衰老模型是国内外比较公认的衰老模型。在一定时间内连续给动物注射D-半乳糖使机体细胞糖代谢及脂代谢紊乱,不仅破坏并消耗机体抗氧化防御系统使自由基积聚,同时还使机体细胞膜脂质受损,过氧化脂质、脂褐素增高,出现衰老。D-半乳糖模型因全面影响细胞的代谢功能和一些重要的酶的功能,所导致的老化较全面。其老化与机能减退具体机制可能与代谢障碍、免疫损伤、自由基损伤及线粒体损伤等相关。为探讨外源性脂肪源干细胞移植是否具有延缓衰老的作用,本实验观察移植ADSCs对D-半乳糖(D-gal)衰老大鼠自由基及免疫等方面及衰老大鼠学习记忆及性能力等方面的影响,探索一种新的抗衰老机制,为临床延缓衰老的治疗提供新的思路和方法。研究移植脂肪源干细胞对D-半乳糖致衰老大鼠相关衰老指标,学习记忆能力及交配能力的影响,进一步为筋膜结缔组织为干细胞的储备源泉提供证据支持,为筋膜学提供理论支撑,并探讨临床替代疗法的治疗机理。 1、判断非特异性筋膜结缔组织中是否存在脂肪源干细胞； 2、观察移植脂肪源干细胞能否对D-半乳糖衰老大鼠自由基及免疫功能产生影响,阐述移植脂肪源干细胞对抗D-半乳糖衰老的血清学意义； 3、通过Morris迷宫,研究移植筋膜结缔组织中的脂肪源干细胞能否对D-半乳糖衰老大鼠学习记忆能力产生影响；通过免疫组织化学染色,观察海马锥体细胞形态学变化,及其凋亡状况。 4、通过交配实验,研究移植筋膜结缔组织中的脂肪源干细胞能否提高D-半乳糖衰老大鼠的性能力；通过免疫组织化学染色,观察移植标记的脂肪源干细胞在睾丸组织中的定位和移位情况,及睾丸间质细胞分泌3p-HSD情况。通过血清学检测,观察睾酮分泌状况。为“筋膜学”抗衰老相关理论提供进一步佐证。 1、取成年大鼠腹股沟脂肪垫,酶消化法分离、培养筋膜结缔组织脂肪源干细胞。通过形态学、功能学、流式细胞术鉴定非特异性筋膜结缔组织源细胞是否符合间充质干细胞的特性,以判断非特异性筋膜结缔组织中是否含有间充质干细胞。 2、90只SD大鼠随机分成空白对照组(A组)、模型组(B组)和治疗组(C组)。B、C组颈背部皮下注射15%D-半乳糖1OOOmg/(kg-d),连续8周复制亚急性衰老模型,空白对照组大鼠每天给予生理盐水1.6ml颈背部皮下注射。 3、造衰老模型成功后,细胞治疗组大鼠行脂肪源干细胞静脉移植。用于移植的第4代ADSCs在进行传代后,在培养基中掺入10μmol/L的Brdu,培养3天后消化收集,无菌生理盐水重悬,300万/ml。每只大鼠经尾静脉注射ADSCs3×106个,模型组输入生理盐水1ml。 4、移植细胞后第10天开始,连续5天对各组大鼠进行Morris水迷宫实验,检测大鼠空间学习记忆能力。HE染色观察大鼠海马区椎体细胞形态,TUNEL免疫组化染色观察大鼠海马区锥体细胞的凋亡指数。 5、雌鼠分别在实验前48h和4h皮下注射苯甲酸雌二醇200μg/kg和黄体酮2mg/kg以诱导雌鼠发情,用发情的雌鼠和雄鼠进行交配,从而对雄性进行相关性能力研究。HE染色显示大鼠睾丸组织精曲小管结构变化,免疫组化及免疫荧光追踪移植细胞；免疫组化观察3β-HSD分泌及大鼠间质细胞凋亡指数。放免法观察大鼠血清睾酮含量。 1、非特异筋膜结缔组织源细胞具有长梭形、多角形的形态学特征,可以形成细胞集落,体外诱导可以分化成成骨细胞、脂肪细胞。 2、连续皮下注射D-gal可以模拟大鼠衰老。移植ADSCs后,可以明显提高衰老大鼠T-SOD、CuZn-SOD活性、血清IL-2水平、脾脏指数和胸腺指数,降低MDA水平(P0.05)；细胞治疗组血清NO水平与模型组比较,无统计学差异(P0.05)。 3、Morris迷宫显示移植干细胞后,衰老大鼠空间学习记忆能力提高；TUNEL染色显示海马锥体细胞凋亡率降低。 4、移植干细胞后同时可以改善衰老大鼠交配能力,增加交配次数和扑捉次数。细胞治疗后血清睾酮含量增加,睾丸间质细胞分泌3β-HSD增多,而间质细胞凋亡率降低。 1、筋膜结缔组织脂肪源干细胞具有长梭形、多角形的形态学特征,可以形成细胞集落,与其它组织来源间充质干细胞无明显差别；细胞表面标记物检测中,4代贴壁ADSCs特异性表面标志物CD90、CD29表达率分别为90%以上,而CD49d、CD11b、CD45阴性表达,而CD106弱表达阳性；该细胞经定向诱导后可以成骨、成脂肪,与间充质干细胞多向分化的功能一致。可以判断在发育成熟大鼠的筋膜结缔组织中存在未分化间充质干细胞。 2、连续颈背部皮下注射D-gal可以有效模拟亚急性衰老模型。衰老大鼠在外观上及自由基等方面,显示明显衰老征象。 3、静脉移植脂肪源干细胞,可以显著升高衰老大鼠体内SOD含量、IL-2水平,有效降低MDA含量,同时提高胸腺和脾脏指数。 4、移植脂肪源干细胞可以明显改善衰老大鼠的空间学习记忆能力,抑制海马锥体细胞凋亡。 5、移植脂肪源干细胞可以改善衰老老鼠的交配能力,促进睾酮释放,同时保护睾丸间质细胞。综上所述,本研究分别采用了细胞分离培养、HE染色和免疫组化等技术手段,从血清学自由基及免疫功能方面,以及空间学习记忆能力、交配能力等多方面,多角度的探讨了移植脂肪源干细胞对D-gal诱导的亚急性衰老大鼠的作用。这是从筋膜学角度研究抗衰老机理的一个新尝试。本研究发现,补充外源性的脂肪源干细胞可以延缓D-gal诱导的衰老,提高其生命质量。从而为筋膜学的支持与储备理论提供实验支持。
[Abstract]:Aging is the decline of the whole body morphology and physiological function, is a dynamic and complex process. The prevention and anti-aging has become a hot topic in life science research. Professor Lin proposed fasciaology hypothesis holds that the human body is not yet differentiated by nonspecific connective tissue (fascia) support and reserve system the composition and function of the system to support differentiated functional cells based on a common form. The function of maintaining the structure and function of the system is stable in the support and reserve system support, support and reserve system to keep the function of the system and function of cell renewal activities provide nutrients Everfount cell source and cell activity needed. Function of the system is based on the function of the cell can be designed on the basis of specialization, and support and reserve system is to support the fascia of stem cells as the core, through the differentiation Stem cells, providing Everfount cells as a function of system updates, and a variety of cell functions of the system update, metabolism provides a stable internal environment. Aging is the process of gradual depletion of stem cell reserve in fascia, and how to maintain the normal state of fascia, provide a stable cell resource and to maintain its the normal differentiation of cells as a function of the system, the key is to extend the life cycle. If the aging reduced when stem cells can be timely added, providing a new cell reserve for the body to repair the body damage, delay aging, prolong the life cycle.
Mesenchymal stem cells (menchymal stem cells) has self-renewal and multilineage differentiation capacity. Initially it was found by Friendenstein, and proves that it can differentiate into osteoblasts and adipocytes in vitro, then the other research team found that bone marrow derived MSCs can not only differentiate into mesodermal tissue, and differentiation for endoderm and ectoderm derived tissues. The systemic distribution of mesenchymal stem tissue and fascia connective tissue cells is consistent, the longitudinal, support and reserve role fascia connective tissue biological characteristics and differentiation of the transverse fascia of the body is consistent with the.ADSCs.ADSCs is a form of stem cell reserve undifferentiated fascia with multiple differentiation potential and can differentiate.ADSCs with low immunogenicity, immune function and cytokine secretion ability for different body movement Plants have provided favorable conditions, make ADSCs become one of the highlights of biological treatment; ADSCs cytokine secretion of anti aging effect, has been widely used in clinical.ADSCs replacement therapy can not only be used in a variety of degenerative diseases and failure, and can be used for immunotherapy of.ADSCs may be aided through the following mechanisms: first tissue repair or reconstruction, ADSCs was implanted in a damaged or necrotic tissue, they can secrete the way to associate the secretion of cytokines and growth factors to stimulate the repair of.ADSCs can provide antioxidants, free radical scavenger, heat shock protein to repair tissue, to remove toxic substances released by the local environment, promote the repair is still the survival of cells. In addition, ADSCs can stimulate endogenous stem cells to repair organs or to supplement its direct transfer added to the area to be repaired, In order to regulate the microenvironment of stem cells and promote them to differentiate into functional cells along the way they need, they renew the aging functional cells and maintain homeostasis.
The subacute aging model induced by D-gal is compared with aging model recognized at home and abroad. In a certain period of time for animal injection of D- galactose in the cells of the body metabolism of glucose and lipid metabolism, not only destroy and consume the antioxidant defense system to free radical accumulation, but also make the cell membrane lipid damage, lipid peroxidation, lipofuscin increased.D- galactose senescence model, due to the overall impact on cell metabolism and function of some important enzymes, which lead to more comprehensive aging. The specific mechanisms of aging and functional decline may be related to metabolic disorders, immune injury, free radical damage and mitochondrial injury.
In order to explore the effect of adipose derived stem cell transplantation has anti-aging effect, the effects of ADSCs transplantation on D- galactose (D-gal) attenuation influence rats free radicals and immune aspects and aging rats learning and memory ability etc., to explore a new anti-aging mechanism, to provide ideas and methods new clinical anti-aging treatment.
Study on transplantation of adipose derived stem cells induced by aging related indexes of aging rats of D- galactose, affect the ability of learning and memory and mating ability, and provide further evidence to support the fascia connective tissue stem cell reserve source, to provide theoretical support for fasciaology, and to explore the clinical replacement therapy treatment mechanism.
1, to determine whether there is a fat source stem cell in the nonspecific fascia connective tissue.
2, to observe whether transplantation of adipose derived stem cells can affect the free radicals and immune function of D- galactose aging rats, and to explain the serological significance of transplantation of adipose derived stem cells against D- galactose senescence.
3, we studied the effects of adipose derived stem cells from transplanted fascial connective tissue on learning and memory ability in D- galactose aging rats through Morris labyrinth. We observed the morphological changes and apoptosis of pyramidal cells in hippocampus by immunohistochemical staining.
4, through mating experiment, transplantation of fascia connective tissue in adipose derived stem cells can improve the aging rats induced by D- galactose ability; immunohistochemical staining was used to observe the transplanted adipose derived stem cells in the testes of localization and translocation, and secretion of Leydig cells by serological 3p-HSD. Detection, observation of testosterone secretion. To provide further evidence for the "fasciaology" anti aging related theory.
1, adult inguinal fat pads of rats were isolated by enzymatic digestion method, cultivation of fascia connective tissue, adipose derived stem cells. By morphology, flow cytometry, identification of fascia connective tissue derived cells is consistent with the characteristics of mesenchymal stem cells, to determine whether the fascia connective tissue contains mesenchymal stem cells.
2,90 SD rats were randomly divided into blank control group (group A), model group (group B) and treatment group (group C).B and C group were subcutaneously injected with 15%D- galactose 1OOOmg/ (kg-d) on the back of the neck. The subacute aging model was replicated for 8 weeks. The blank control group was given saline every day, and the neck and back were subcutaneously injected.
3, made after the success of the aging model, cell therapy group rats underwent intravenous transplantation of adipose derived stem cells for transplantation. The fourth generation of ADSCs in the passage, in the cultivation of 10 mol/L mixed medium Brdu, collected 3 days after cultured by digestion, sterile physiological saline, 3 million /ml. per rat by the tail intravenous injection of ADSCs3 * 106, 1ml. normal saline model group input
4, after tenth days of transplantation, 5 days in each group, rats in each group were subjected to Morris water maze test for 5 days. The spatial learning and memory ability of rats was detected..HE staining was used to observe the morphology of hippocampal vertebral cells. The apoptotic index of pyramidal cells in hippocampus of rats was observed by immunohistochemical staining.
5, female rats respectively before the experiment 48h and 4H subcutaneous injection of estradiol benzoate and progesterone 200 g/kg 2mg/kg female rats to induce estrus, with estrous female rats and male rats were mated to study the relationship between.HE staining of rat testis seminiferous tubule structure of male, immunohistochemistry immunofluorescence and tracking of transplanted cells; immunohistochemistry 3 beta -HSD secretion and rat Leydig cell apoptosis index. Radioimmunoassay method was used to observe the testosterone content in serum of rats.
1, nonspecific fascial connective tissue derived cells have long spindle shape and polygonal morphology. They can form cell colonies, and can differentiate into osteoblasts and adipocytes in vitro.
2, continuous subcutaneous injection of D-gal can be simulated in aging rats. After transplantation of ADSCs can significantly improve T-SOD, CuZn-SOD activity of aging rats, serum IL-2 levels, spleen index and thymus index, reduce the level of MDA (P0.05) cells; the serum NO level of the treatment group compared with model group, no statistical difference (P0.05).
3, after the Morris labyrinth showed the transplanted stem cells, the spatial learning and memory ability of the aged rats increased, and the TUNEL staining showed that the apoptosis rate of hippocampal pyramidal cells decreased.
4, transplantation of stem cells can improve mating ability, increase mating times and catch times in aging rats. After treatment, serum testosterone content increased, testicular interstitial cells secreted 3 beta -HSD and interstitial cell apoptosis rate decreased.
1, the fascia connective tissue, adipose derived stem cells have long fusiform, polygonal morphology, can form cell colonies, with no significant difference in other tissue derived mesenchymal stem cells; cell surface markers, the 4 generation of adherent ADSCs specific surface markers CD90, CD29 expression rate was above 90% and, CD49d, CD11b, CD45 and CD106 negative expression, weak positive expression; the cells were induced to osteoblasts, adipocytes, consistent with the function of mesenchymal stem cell differentiation. One can determine in fascia connective tissue maturation of rats in the presence of undifferentiated mesenchymal stem cells.
2, subcutaneous injection of D-gal on the back of the neck of the continuous neck can effectively simulate the subacute aging model. The old rats have obvious signs of aging in the appearance and free radicals.
3, intravenous transplantation of adipose derived stem cells could significantly increase SOD content, IL-2 level, reduce MDA content and increase the thymus and spleen index.
4, transplantation of adipose derived stem cells can obviously improve the spatial learning and memory ability of aging rats and inhibit the apoptosis of hippocampal pyramidal cells.
5, transplantation of fat derived stem cells can improve the mating ability of aging mice, promote the release of testosterone, and protect the Leydig cells.
In summary, this study adopted the cell culture, HE staining and immunohistochemical techniques from free radicals and immune function in serum, and the ability of spatial learning and memory ability and other aspects, mating, multi angle of transplantation of adipose derived stem cells on subacute aging rats induced by D-gal. This is a new attempt from the Perspective of the anti-aging mechanism of fascia. The study found that exogenous stem cells can delay D-gal induced senescence, improve their quality of life. In order to provide experimental support for the preparation and storage support fasciaology theory.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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