發(fā)布時間：2018-01-12 21:34

  本文關(guān)鍵詞：移植脂肪源干細胞對D-半乳糖衰老大鼠的影響　出處：《南方醫(yī)科大學》2010年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 衰老 脂肪源干細胞 筋膜學 SOD MDA NO IL-2 睪酮

【摘要】： 衰老是整個機體形態(tài)結(jié)構(gòu)和生理機能的全面衰退,是一個動態(tài)的、復雜的過程。當前預防和延緩衰老已成為生命科學研究的熱點。原林教授提出的“筋膜學假說”認為人體是由尚未分化的非特異性結(jié)締組織(筋膜)支架所構(gòu)成的支持與儲備系統(tǒng)和以已分化功能細胞為基礎的功能系統(tǒng)所共同構(gòu)成。功能系統(tǒng)在支持與儲備系統(tǒng)支持下維持結(jié)構(gòu)與功能的穩(wěn)定,支持與儲備系統(tǒng)不斷的為功能系統(tǒng)的細胞更新和功能活動提供源源不斷的細胞源泉和細胞活動所需要的營養(yǎng)物質(zhì)。功能系統(tǒng)是建立在功能細胞的專能特化基礎上的,而支持與儲備系統(tǒng)的筋膜支架是以干細胞為核心,通過分化出各種定向干細胞,為功能系統(tǒng)的更新提供源源不斷的細胞補充,并為功能系統(tǒng)的各種細胞的更新、代謝提供一個穩(wěn)定的內(nèi)部環(huán)境。衰老是筋膜中干細胞儲備逐漸耗竭的過程,而如何保持筋膜的正常狀態(tài),為功能系統(tǒng)提供穩(wěn)定的細胞來源并維持其向功能細胞的正常分化,是延長生命周期的關(guān)鍵。如果衰老時減少的干細胞能得到及時的補充,為機體提供新的細胞儲備,可修復機體損傷,延緩衰老、延長生命周期。 間充質(zhì)干細胞(menchymal stem cells)具有自我更新、多向分化的能力。最初是由Friendenstein等發(fā)現(xiàn),且證明其在體外可以分化成為成骨細胞及脂肪細胞,而后其它的研究小組發(fā)現(xiàn)骨髓來源的MSCs不僅可以分化為中胚層來源的組織,而且可以分化為內(nèi)胚層和外胚層來源的組織。間充質(zhì)干細胞的多組織來源與筋膜結(jié)締組織支架的全身分布是一致的,其縱向、橫向分化的生物學特性與筋膜學提出的筋膜結(jié)締組織對機體的支持儲備作用也是一致的。ADSCs是筋膜中未分化干細胞的一種儲備形式。ADSCs具有多分化潛能,能夠跨胚層分化。ADSCs具有低免疫原性,免疫調(diào)節(jié)功能和分泌細胞因子等能力為異體移植提供了有利條件,使ADSCs成為生物治療的亮點之一；ADSCs分泌細胞因子抗老化的作用,已經(jīng)被臨床廣泛應用。ADSCs不僅可以用來進行各種退行性和衰竭疑難病的替代治療,而且可用來進行輔助的免疫治療。ADSCs可能通過以下機制修復或者重建組織：首先,ADSCs被植入一個受損的或是壞死的組織,它們能以副分泌的方式分泌出細胞活素和生長因子以刺激修復。ADSCs可以提供抗氧化劑、自由基清除劑、熱休克蛋白等給待修復組織,從而清除局部環(huán)境中所釋放的有毒物質(zhì),促進仍存活的細胞的修復。另外,ADSCs可以通過刺激內(nèi)生性干細胞補充到待修復的臟器或自身直接遷移補充到待修復區(qū)域,以調(diào)節(jié)干細胞的微環(huán)境,并且促進它們沿著需要的方式分化為功能細胞,從而更新機體衰老的功能細胞,維持機體平衡。 D-gal所致亞急性衰老模型是國內(nèi)外比較公認的衰老模型。在一定時間內(nèi)連續(xù)給動物注射D-半乳糖使機體細胞糖代謝及脂代謝紊亂,不僅破壞并消耗機體抗氧化防御系統(tǒng)使自由基積聚,同時還使機體細胞膜脂質(zhì)受損,過氧化脂質(zhì)、脂褐素增高,出現(xiàn)衰老。D-半乳糖模型因全面影響細胞的代謝功能和一些重要的酶的功能,所導致的老化較全面。其老化與機能減退具體機制可能與代謝障礙、免疫損傷、自由基損傷及線粒體損傷等相關(guān)。 為探討外源性脂肪源干細胞移植是否具有延緩衰老的作用,本實驗觀察移植ADSCs對D-半乳糖(D-gal)衰老大鼠自由基及免疫等方面及衰老大鼠學習記憶及性能力等方面的影響,探索一種新的抗衰老機制,為臨床延緩衰老的治療提供新的思路和方法。 研究移植脂肪源干細胞對D-半乳糖致衰老大鼠相關(guān)衰老指標,學習記憶能力及交配能力的影響,進一步為筋膜結(jié)締組織為干細胞的儲備源泉提供證據(jù)支持,為筋膜學提供理論支撐,并探討臨床替代療法的治療機理。 1、判斷非特異性筋膜結(jié)締組織中是否存在脂肪源干細胞； 2、觀察移植脂肪源干細胞能否對D-半乳糖衰老大鼠自由基及免疫功能產(chǎn)生影響,闡述移植脂肪源干細胞對抗D-半乳糖衰老的血清學意義； 3、通過Morris迷宮,研究移植筋膜結(jié)締組織中的脂肪源干細胞能否對D-半乳糖衰老大鼠學習記憶能力產(chǎn)生影響；通過免疫組織化學染色,觀察海馬錐體細胞形態(tài)學變化,及其凋亡狀況。 4、通過交配實驗,研究移植筋膜結(jié)締組織中的脂肪源干細胞能否提高D-半乳糖衰老大鼠的性能力；通過免疫組織化學染色,觀察移植標記的脂肪源干細胞在睪丸組織中的定位和移位情況,及睪丸間質(zhì)細胞分泌3p-HSD情況。通過血清學檢測,觀察睪酮分泌狀況。為“筋膜學”抗衰老相關(guān)理論提供進一步佐證。 1、取成年大鼠腹股溝脂肪墊,酶消化法分離、培養(yǎng)筋膜結(jié)締組織脂肪源干細胞。通過形態(tài)學、功能學、流式細胞術(shù)鑒定非特異性筋膜結(jié)締組織源細胞是否符合間充質(zhì)干細胞的特性,以判斷非特異性筋膜結(jié)締組織中是否含有間充質(zhì)干細胞。 2、90只SD大鼠隨機分成空白對照組(A組)、模型組(B組)和治療組(C組)。B、C組頸背部皮下注射15%D-半乳糖1OOOmg/(kg-d),連續(xù)8周復制亞急性衰老模型,空白對照組大鼠每天給予生理鹽水1.6ml頸背部皮下注射。 3、造衰老模型成功后,細胞治療組大鼠行脂肪源干細胞靜脈移植。用于移植的第4代ADSCs在進行傳代后,在培養(yǎng)基中摻入10μmol/L的Brdu,培養(yǎng)3天后消化收集,無菌生理鹽水重懸,300萬/ml。每只大鼠經(jīng)尾靜脈注射ADSCs3×106個,模型組輸入生理鹽水1ml。 4、移植細胞后第10天開始,連續(xù)5天對各組大鼠進行Morris水迷宮實驗,檢測大鼠空間學習記憶能力。HE染色觀察大鼠海馬區(qū)椎體細胞形態(tài),TUNEL免疫組化染色觀察大鼠海馬區(qū)錐體細胞的凋亡指數(shù)。 5、雌鼠分別在實驗前48h和4h皮下注射苯甲酸雌二醇200μg/kg和黃體酮2mg/kg以誘導雌鼠發(fā)情,用發(fā)情的雌鼠和雄鼠進行交配,從而對雄性進行相關(guān)性能力研究。HE染色顯示大鼠睪丸組織精曲小管結(jié)構(gòu)變化,免疫組化及免疫熒光追蹤移植細胞；免疫組化觀察3β-HSD分泌及大鼠間質(zhì)細胞凋亡指數(shù)。放免法觀察大鼠血清睪酮含量。 1、非特異筋膜結(jié)締組織源細胞具有長梭形、多角形的形態(tài)學特征,可以形成細胞集落,體外誘導可以分化成成骨細胞、脂肪細胞。 2、連續(xù)皮下注射D-gal可以模擬大鼠衰老。移植ADSCs后,可以明顯提高衰老大鼠T-SOD、CuZn-SOD活性、血清IL-2水平、脾臟指數(shù)和胸腺指數(shù),降低MDA水平(P0.05)；細胞治療組血清NO水平與模型組比較,無統(tǒng)計學差異(P0.05)。 3、Morris迷宮顯示移植干細胞后,衰老大鼠空間學習記憶能力提高；TUNEL染色顯示海馬錐體細胞凋亡率降低。 4、移植干細胞后同時可以改善衰老大鼠交配能力,增加交配次數(shù)和撲捉次數(shù)。細胞治療后血清睪酮含量增加,睪丸間質(zhì)細胞分泌3β-HSD增多,而間質(zhì)細胞凋亡率降低。 1、筋膜結(jié)締組織脂肪源干細胞具有長梭形、多角形的形態(tài)學特征,可以形成細胞集落,與其它組織來源間充質(zhì)干細胞無明顯差別；細胞表面標記物檢測中,4代貼壁ADSCs特異性表面標志物CD90、CD29表達率分別為90%以上,而CD49d、CD11b、CD45陰性表達,而CD106弱表達陽性；該細胞經(jīng)定向誘導后可以成骨、成脂肪,與間充質(zhì)干細胞多向分化的功能一致�？梢耘袛嘣诎l(fā)育成熟大鼠的筋膜結(jié)締組織中存在未分化間充質(zhì)干細胞。 2、連續(xù)頸背部皮下注射D-gal可以有效模擬亞急性衰老模型。衰老大鼠在外觀上及自由基等方面,顯示明顯衰老征象。 3、靜脈移植脂肪源干細胞,可以顯著升高衰老大鼠體內(nèi)SOD含量、IL-2水平,有效降低MDA含量,同時提高胸腺和脾臟指數(shù)。 4、移植脂肪源干細胞可以明顯改善衰老大鼠的空間學習記憶能力,抑制海馬錐體細胞凋亡。 5、移植脂肪源干細胞可以改善衰老老鼠的交配能力,促進睪酮釋放,同時保護睪丸間質(zhì)細胞。 綜上所述,本研究分別采用了細胞分離培養(yǎng)、HE染色和免疫組化等技術(shù)手段,從血清學自由基及免疫功能方面,以及空間學習記憶能力、交配能力等多方面,多角度的探討了移植脂肪源干細胞對D-gal誘導的亞急性衰老大鼠的作用。這是從筋膜學角度研究抗衰老機理的一個新嘗試。本研究發(fā)現(xiàn),補充外源性的脂肪源干細胞可以延緩D-gal誘導的衰老,提高其生命質(zhì)量。從而為筋膜學的支持與儲備理論提供實驗支持。
[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.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

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