本文選題：SRC-3 + 基因敲除小鼠　； 參考：《第三軍醫(yī)大學(xué)》2013年博士論文

【摘要】：造血組織對(duì)電離輻射極為敏感，是輻射損傷的主要靶器官之一。輻射射線(xiàn)可導(dǎo)致造血細(xì)胞大量凋亡或死亡，使細(xì)胞周期改變，細(xì)胞增殖遲緩，導(dǎo)致機(jī)體出現(xiàn)嚴(yán)重造血功能障礙。與此同時(shí)，電離輻射還可降低骨髓中基質(zhì)細(xì)胞的數(shù)目和分泌細(xì)胞因子的能力，進(jìn)一步阻礙輻射后造血機(jī)能的恢復(fù)。因此，盡量保護(hù)殘存造血細(xì)胞的數(shù)量、促進(jìn)照后造血細(xì)胞的增殖和維持照后造血微環(huán)境的穩(wěn)定，是治療輻射后造血損傷的有效措施。而如何同時(shí)在造血細(xì)胞和基質(zhì)細(xì)胞中實(shí)現(xiàn)多途徑、多靶點(diǎn)的輻射后保護(hù)，是治療急性放射病造血損傷時(shí)新的思考。 類(lèi)固醇受體輔激活因子-3(steroid receptor coactivator-3, SRC-3)是SRC家族中重要的一員，能夠與多種核受體和轉(zhuǎn)錄因子相互作用，參與激活多條信號(hào)通路，完成相應(yīng)靶基因的轉(zhuǎn)錄。SRC-3不僅能在機(jī)體中發(fā)揮了廣泛的生物學(xué)功能；更作為促瘤因子參與促進(jìn)多種腫瘤細(xì)胞的增殖和生長(zhǎng)，以及對(duì)抗化療藥物引起的凋亡。已有報(bào)道證實(shí)，SRC-3在惡性血液細(xì)胞中高表達(dá)，并能發(fā)揮促增殖和對(duì)抗藥物所致凋亡的作用。因此我們推斷，SRC-3可能在外因所致的造血細(xì)胞損傷中發(fā)揮一定作用。目前，對(duì)SRC-3參與體內(nèi)造血調(diào)控的機(jī)制尚不清楚，關(guān)于在輻射所致造血損傷中的作用更是未見(jiàn)報(bào)道。為了驗(yàn)證SRC-3是否參與了小鼠輻射后造血損傷效應(yīng)，并探明其相關(guān)機(jī)制，我們以SRC-3基因敲除型和野生型小鼠為實(shí)驗(yàn)對(duì)象開(kāi)展了以下實(shí)驗(yàn)。 我們通過(guò)海外合作的方式從美國(guó)休斯敦貝勒醫(yī)學(xué)院引進(jìn)SRC-3基因敲除小鼠的親代小鼠，并在成功繁殖和進(jìn)行基因表型鑒定后，獲得一批理想的SRC-3基因敲除型和野生型實(shí)驗(yàn)用小鼠。在對(duì)所得實(shí)驗(yàn)小鼠按照不同基因型分組后，我們以60Co γ射線(xiàn)分別進(jìn)行了6.0Gy和4.5Gy的全身照射(total body irradiation,TBI)，制造了致死劑量照后和亞致死劑量照后兩種不同的動(dòng)物輻射損傷模型。 為了明確SRC-3在小鼠電離輻射所致造血損傷中的效應(yīng)及探討其相關(guān)機(jī)制，我們分別從體內(nèi)、體外兩方面展開(kāi)實(shí)驗(yàn)。首先，我們?cè)u(píng)估了SRC-3~(-/-)小鼠和野生型小鼠在輻射后不同的造血損傷效應(yīng)，包括整體效應(yīng)，如小鼠一般情況、死亡率、體重等指標(biāo)；外周血細(xì)胞、骨髓造血組織、胸腺和脾臟淋巴組織的損傷情況；并檢測(cè)了小鼠血清中造血相關(guān)細(xì)胞因子（IGF-1，IL-3，IL-6, TPO）的水平變化，驗(yàn)證了SRC-3在小鼠體內(nèi)輻射后造血損傷和造血恢復(fù)過(guò)程中均能發(fā)揮防護(hù)作用。其次，我們以小鼠細(xì)胞作為實(shí)驗(yàn)對(duì)象，分兩部分探討了SRC-3參與調(diào)控和保護(hù)小鼠輻射后造血損傷的機(jī)制：1)以?xún)煞N不同基因型小鼠的骨髓有核細(xì)胞(bone marrow nucleated cells, BMNCs)作為研究對(duì)象，在輻射前和輻射后各時(shí)相點(diǎn)，檢測(cè)了SRC-3對(duì)小鼠BMNCs細(xì)胞的凋亡、增殖、細(xì)胞周期、相關(guān)增殖通路的影響，以及對(duì)凋亡相關(guān)分子(NF-κB,p53,Bcl-2,Bax)和周期調(diào)控因子(cyclin A，E, D1，CDK2，CDK4，p21,p53)的調(diào)控作用，闡明了SRC-3在輻射后的小鼠BMNCs中的調(diào)控和保護(hù)作用。2)以?xún)煞N不同基因型小鼠的骨髓基質(zhì)細(xì)胞(bone marrow stromal cells, BMSCs)作為研究對(duì)象，在輻射前和輻射后各時(shí)相點(diǎn)，檢測(cè)了SRC-3對(duì)小鼠BMSCs細(xì)胞的增殖活性、成纖維細(xì)胞集落形成單位(colony-forming unit of fibroblast, CFU-F)形成能力、增殖相關(guān)p-AKT蛋白表達(dá)量，和上清中VCAM-1，IGF-1，IL-3，IL-6和TPO水平的影響，明確了SRC-3在小鼠BMSCs細(xì)胞中的調(diào)控作用。所得結(jié)果總結(jié)如下： 主要結(jié)果： 1.以SRC-3+/-雜合子小鼠為親代交配繁殖得到一定數(shù)目的野生型(SRC-3+/+)、雜合子(SRC-3+/-)和基因敲除的純合子(SRC-3~(-/-))小鼠。通過(guò)PCR和Western blot方法分別對(duì)子代小鼠進(jìn)行基因表型和蛋白表型的鑒定后，將所得SRC-3~(-/-)小鼠SRC-3+/+小鼠分組供實(shí)驗(yàn)用。蛋白表型鑒定結(jié)果發(fā)現(xiàn)SRC-3~(-/-)小鼠中SRC-3基因已經(jīng)徹底敲除，其骨髓、脾臟和胸腺組織中未測(cè)得SRC-3蛋白的表達(dá)；而野生型小鼠中骨髓細(xì)胞的SRC-3蛋白的表達(dá)水平顯著高于其在脾臟和胸腺中的表達(dá)水平(P0.05)。 2. SRC-3~(-/-)小鼠與同齡野生型小鼠比較，具有體重輕、身材短小、發(fā)育遲緩，成年雌鼠性成熟障礙，不易受孕等特點(diǎn)。正常情況下，與野生型比較，SRC-3~(-/-)小鼠體重和血清中IGF-1水平顯著偏低，兩者比較存在統(tǒng)計(jì)學(xué)差異(P0.05)。并且發(fā)現(xiàn)，SRC-3~(-/-)小鼠的外周血中WBC、RBC和PLT計(jì)數(shù)和骨髓有核細(xì)胞計(jì)數(shù)均略低于野生型；胸腺指數(shù)、脾臟指數(shù)略高于野生型，但兩者比較無(wú)統(tǒng)計(jì)學(xué)差異。 3.給予致死劑量TBI后，SRC-3~(-/-)小鼠顯現(xiàn)出更嚴(yán)重的整體損傷效應(yīng)，表現(xiàn)為一般情況更差、體重下降明顯，30天生存率更低，與野生型比較存在統(tǒng)計(jì)學(xué)差異(P0.05)，提示SRC-3~(-/-)小鼠機(jī)體抗輻射能力更弱。 4.給予亞致死劑量TBI后，SRC-3~(-/-)小鼠中外周血血象更低，骨髓有核細(xì)胞計(jì)數(shù)和造血集落形成單位計(jì)數(shù)更少，與野生型比較存在統(tǒng)計(jì)學(xué)差異(P0.05)。同時(shí)骨髓病理切片提示所見(jiàn)輻射后SRC-3~(-/-)小鼠的骨髓更空曠。提示SRC-3~(-/-)小鼠輻射后顯現(xiàn)出更加嚴(yán)重的外周血和骨髓的造血損傷效應(yīng)。值得一提的是，SRC-3~(-/-)小鼠的巨核系造血細(xì)胞在輻射后損傷尤重，在照射后11天極低點(diǎn)至隨后恢復(fù)過(guò)程中的各時(shí)相點(diǎn)，均可觀(guān)察到SRC-3~(-/-)小鼠的外周血PLT計(jì)數(shù)、病理切片中骨髓腔巨核細(xì)胞數(shù)目和體外培養(yǎng)巨核系集落形成單位顯著低于野生型，兩者比較存在統(tǒng)計(jì)學(xué)差異(P0.05)。 5.給予亞致死劑量TBI后，在各個(gè)時(shí)相點(diǎn)可觀(guān)察到，SRC-3~(-/-)小鼠的脾臟指數(shù)和胸腺指數(shù)均略高于野生型，并與野生型比較無(wú)統(tǒng)計(jì)學(xué)差異。說(shuō)明SRC-3~(-/-)小鼠脾臟和胸腺的輻射后損傷程度明顯低于骨髓損傷程度，因此SRC-3~(-/-)小鼠的淋巴組織在輻射后的損傷效應(yīng)與骨髓組織有所不同。 6.給予亞致死劑量TBI后，SRC-3~(-/-)小鼠血清中IGF-1水平在照后各時(shí)相點(diǎn)持續(xù)處于較低水平，與野生型比較存在統(tǒng)計(jì)學(xué)差異(P0.05)。SRC-3~(-/-)小鼠血清中造血因子IL-3和IL-6在照后第7天顯著均低于野生型，兩者比較存在統(tǒng)計(jì)學(xué)差異(P0.05)。血清中細(xì)胞因子TPO水平則未發(fā)現(xiàn)存在組間差異。 7.用流式法測(cè)定兩組小鼠的BMNCs的凋亡率和所含Sca-1+細(xì)胞比例后發(fā)現(xiàn)，在正常情況下，兩種小鼠BMNCs的凋亡率和所含Sca-1+細(xì)胞比例無(wú)統(tǒng)計(jì)學(xué)差異。而輻射后SRC-3~(-/-)小鼠的BMNCs中凋亡率顯著升高和Sca-1+細(xì)胞比例顯著降低，與野生型比較存在統(tǒng)計(jì)學(xué)差異(P0.05)。同時(shí)，用Western blot方法測(cè)得輻射后SRC-3~(-/-)小鼠BMNCs中的凋亡蛋白p53和Bax表達(dá)顯著升高，抗凋亡蛋白NF-κB (p65)和Bcl-2表達(dá)顯著降低，與野生型比較存在統(tǒng)計(jì)學(xué)差異(P0.05)。提示SRC-3~(-/-)小鼠的BMNCs在輻射后凋亡增多、殘留數(shù)目較少；其增高的凋亡敏感性與凋亡相關(guān)蛋白的表達(dá)量異常有關(guān)。 8.用流式法測(cè)定兩組小鼠的BMNCs的細(xì)胞周期和增殖指數(shù)后發(fā)現(xiàn)，在正常情況下，兩種小鼠BMNCs的細(xì)胞周期分布和增殖指數(shù)無(wú)統(tǒng)計(jì)學(xué)差異。而輻射后SRC-3~(-/-)小鼠的BMNCs表現(xiàn)出G0/G1期顯著升高，S期阻滯的特點(diǎn)，同時(shí)增殖指數(shù)也顯著低于野生型(P0.05)。進(jìn)一步研究發(fā)現(xiàn)，輻射后SRC-3~(-/-)小鼠BMNCs中正性周期調(diào)控因子，包括CyclinD1、CyclinE、 CyclinA、CDK2、CDK4的表達(dá)水平顯著降低；負(fù)性周期調(diào)控因子p53和p21的蛋白表達(dá)量顯著升高，與野生型比較有統(tǒng)計(jì)學(xué)差異(P0.05)。并且，SRC-3~(-/-)小鼠BMNCs中p-AKT蛋白表達(dá)量在輻射后下降，表明SRC-3~(-/-)小鼠AKT增殖信號(hào)通路在輻射后活性降低。以上結(jié)果提示SRC-3~(-/-)小鼠的BMNCs在輻射后存在細(xì)胞周期分布異常和增殖障礙的特點(diǎn)，其中周期調(diào)控因子的表達(dá)水平和的AKT信號(hào)通路活性輻射后降低，是導(dǎo)致輻射后造血恢復(fù)緩慢的重要原因。 9.用CKK-8法測(cè)定體外培養(yǎng)所得小鼠BMSCs的增殖活性后發(fā)現(xiàn)，正常情況下，SRC-3~(-/-)小鼠的增殖活性顯著低于野生型(P0.05)；而輻射后兩者組間差異更加顯著(P0.01)。并且，SRC-3~(-/-)小鼠的CFU-F形成能力在照前和照后均顯著低于野生型，兩者比較有統(tǒng)計(jì)學(xué)差異(P0.05)。另外，我們還檢測(cè)了小鼠BMSCs中調(diào)控增殖的AKT信號(hào)通路活性，發(fā)現(xiàn)照前兩組小鼠BMSCs中p-AKT蛋白表達(dá)量水平接近；但在照后SRC-3~(-/-)小鼠BMSCs中p-AKT蛋白表達(dá)量顯著低于野生型，兩組比較有統(tǒng)計(jì)學(xué)差異(P0.05)。以上結(jié)果說(shuō)明SRC-3~(-/-)小鼠BMSCs的增殖能力和CFU-F形成能力在照射前降低，輻射后程度加重；而照射后SRC-3~(-/-)小鼠BMSCs中AKT增殖信號(hào)通路活性降低可能是導(dǎo)致該損害加重的一個(gè)重要原因。 10.用ELISA法測(cè)定小鼠BMSCs細(xì)胞培養(yǎng)上清液中的細(xì)胞因子水平后發(fā)現(xiàn)，正常情況下，SRC-3~(-/-)小鼠BMSCs上清中VCAM-1和IGF-1水平偏低，與野生型比較存在統(tǒng)計(jì)學(xué)差異(P0.05)。而輻射損傷后，SRC-3~(-/-)小鼠BMSCs上清中VCAM-1、IGF-1、IL-3和IL-6水平均顯著低于野生型，兩者比較具有統(tǒng)計(jì)學(xué)差異(P0.05)，說(shuō)明SRC-3~(-/-)小鼠BMSCs分泌細(xì)胞因子的能力顯著低于野生型。 結(jié)論： 1.缺乏SRC-3可增加致死劑量TBI后小鼠死亡率，表明SRC-3可以影響輻射后小鼠的存活率，，在整體水平上提高小鼠對(duì)輻射損傷的防護(hù)能力。 2.缺乏SRC-3可導(dǎo)致亞致死劑量TBI后，小鼠骨髓損傷更重、造血恢復(fù)更緩慢。因此，SRC-3能減輕小鼠輻射后骨髓造血損傷程度，促進(jìn)造血恢復(fù)，特別是對(duì)巨核系造血影響更加明顯。 3. SRC-3與維持血清中IGF-1、IL-3和IL-6水平有密切關(guān)系。缺乏SRC-3可導(dǎo)致照射前后血清中的細(xì)胞因子IGF-1顯著降低，以及照射后血清中IL-3和IL-6水平嚴(yán)重不足。 4. SRC-3可保護(hù)輻射后小鼠BMNCs中存活的Sca-1+細(xì)胞和降低凋亡細(xì)胞，并通過(guò)激活NF-κB (p65)蛋白和抑制p53蛋白輻射后的活性，降低小鼠BMNCs輻射后凋亡敏感性。 5. SRC-3可促進(jìn)輻射后小鼠BMNCs的增殖和周期進(jìn)程，降低G0/G1期比例，減少S期阻滯。其機(jī)制與上調(diào)正性周期調(diào)控因子CyclinD1、CyclinE、 CyclinA、CDK2、CDK4和下調(diào)負(fù)性周期調(diào)控因子p21和p53的表達(dá)水平，激活A(yù)KT信號(hào)通路有關(guān)。 6.缺乏SRC-3可導(dǎo)致正常情況下小鼠BMSCs的增殖活性、CFU-F形成能力，以及BMSCs上清中VCAM-1和IGF-1的水平顯著下降。不僅如此，還可降低輻射后小鼠BMSCs中的p-AKT蛋白表達(dá)和上清中IL-3和IL-6的水平。因此SRC-3促進(jìn)基質(zhì)細(xì)胞的增殖和分泌細(xì)胞因子的能力；通過(guò)調(diào)控造輻射后基質(zhì)細(xì)胞的數(shù)目和細(xì)胞因子的水平，維護(hù)造血微環(huán)境的穩(wěn)定來(lái)實(shí)現(xiàn)對(duì)輻射后造血的防護(hù)。
[Abstract]:The hematopoietic tissue is very sensitive to ionizing radiation, which is one of the main target organs of radiation damage. Radiation rays can cause a large number of apoptosis or death of hematopoietic cells, change the cell cycle and slow the proliferation of cells, and cause serious hematopoietic dysfunction. At the same time, ionizing radiation can also reduce the number and secretion of stromal cells in the bone marrow. The ability of cell factors further hinders the recovery of hematopoietic function after radiation. Therefore, it is an effective measure to protect the number of residual hematopoietic cells, promote the proliferation of hematopoietic cells after irradiation and maintain the stability of the hematopoietic microenvironment after irradiation, which is an effective measure for the treatment of hematopoietic injury after radiation. The radiation protection after target is a new thinking for treating hematopoietic injury of acute radiation sickness.
The steroid receptor coactivator -3 (steroid receptor coactivator-3, SRC-3) is an important member of the SRC family. It can interact with a variety of nuclear receptors and transcription factors and participate in activating multiple signal pathways. The transcriptional.SRC-3 of the corresponding target gene can not only play a wide biological function in the body, but also act as a tumor stimulating factor. It has been reported that SRC-3 is highly expressed in malignant blood cells and can play a role in promoting proliferation and antagonism to drug induced apoptosis. Therefore, we infer that SRC-3 may play a role in the injury of hematopoietic cells caused by external causes. The mechanism of SRC-3's involvement in the regulation of hematopoiesis in vivo is not clear. There is no report about the role of the hematopoiesis in radiation induced hematopoiesis. In order to verify whether SRC-3 participates in the effect of postradiation hematopoiesis in mice, and the related mechanisms are explored, the following experiments have been carried out on SRC-3 gene knockout and wild type mice.
We introduced SRC-3 gene knockout mice from the Baylor College of Medicine, Houston, in the way of overseas cooperation. After successful reproduction and genetic phenotype identification, a group of ideal SRC-3 knockout and wild type experimental mice were obtained. After grouping the experimental mice in accordance with different genotypes, we used 60Co The total body radiation of 6.0Gy and 4.5Gy (total body irradiation, TBI) was carried out by gamma ray, and two different animal radiation damage models were made after lethal dose illumination and sublethal dose irradiation.
In order to identify the effect of SRC-3 on the hematopoietic damage induced by ionizing radiation in mice and to explore its related mechanisms, we carried out the experiment from two aspects in vivo and in vitro. First, we evaluated the effects of different hematopoiesis in SRC-3~ (- / -) mice and wild type mice after radiation, including the overall effect, such as the general condition, mortality and weight of mice. The damage of peripheral blood cells, bone marrow hematopoietic tissue, thymus and spleen lymphatic tissue, and the level changes of hematopoiesis related cytokines (IGF-1, IL-3, IL-6, TPO) in the serum of mice were detected, and the protective effect of SRC-3 on hematopoiesis and recovery of blood after radiation in mice was verified. Secondly, we were small. As the experimental object, the mouse cells were divided into two parts to explore the mechanism of SRC-3 participation in regulating and protecting the hematopoiesis after radiation in mice: 1) the bone marrow nucleated cells (bone marrow nucleated cells, BMNCs) of two different genotypes mice were used as the research object. The SRC-3 effect on the death of BMNCs cells in mice before and after radiation was detected. The effects of apoptosis related molecules (NF- kappa B, p53, Bcl-2, Bax) and cyclical regulators (cyclin A, E, D1, CDK2, CDK4, p21), as well as the regulation and protection of the two different genotypic mice Marrow stromal cells, BMSCs), as the research object, detected the proliferation activity of SRC-3 on mouse BMSCs cells before and after radiation, and the formation of colony forming unit of fibroblasts (colony-forming unit of fibroblast, CFU-F). The effect of Ping on the regulation of SRC-3 in mouse BMSCs cells was clarified.
Main results:
1. a certain number of wild type (SRC-3+/+), heterozygote (SRC-3+/-) and gene knockout homozygote (SRC-3~ (/ -)) mice were obtained by parental mating of SRC-3+/- heterozygote mice. After identification of gene phenotypes and protein forms of offspring mice by PCR and Western blot, the SRC-3+/+ mice were divided into SRC-3~ (- / -) mice. The results of protein phenotypic identification found that the SRC-3 gene in SRC-3~ (- / -) mice had been completely knocked out, and the expression of SRC-3 protein was not detected in the bone marrow, spleen and thymus tissues, while the expression level of SRC-3 protein in the bone marrow cells in the wild type mice was significantly higher than that in the spleen and thymus (P0.05).
2. SRC-3~ (- / -) mice, compared with the same age wild type mice, have the characteristics of light weight, short stature, retardation, adult female sexual maturity and not easily conceived. Under normal conditions, the weight of SRC-3~ (- / -) mice and the IGF-1 level in serum were significantly lower than those in the wild type, and there were statistical differences (P0.05). And SRC-3~ (- (-)) The number of WBC, RBC, PLT and bone marrow nucleated cells in the peripheral blood of the mice were slightly lower than that in the wild type, and the thymus index and spleen index were slightly higher than those of the wild type, but there was no statistical difference between them.
3. after the lethal dose of TBI, the SRC-3~ (- / -) mice showed a more severe overall damage effect, showing a worse general condition, a significant decrease in weight, a lower 30 natural survival rate, and a statistical difference from the wild type (P0.05), suggesting that the anti radiation ability of the SRC-3~ (- / -) mice was weaker.
4. after the sublethal dose of TBI, the peripheral blood hemogram of the SRC-3~ (- / -) mice was lower, the count of nucleated cells in the bone marrow and the number of the hematopoietic colony forming units were less, and there was a statistical difference between the wild type and the wild type (P0.05). Meanwhile, the bone marrow pathological sections suggested that the bone marrow of the SRC-3~ (- / -) mice after the radiation was more open. It suggested that the SRC-3~ (- / -) mice were irradiated after the radiation. It is worth mentioning that the SRC-3~ (- / -) megakaryocyte hematopoietic cells are particularly damaged after radiation, and the PLT count of the peripheral blood of SRC-3~ (- / -) rats can be observed at 11 days after irradiation, and the marrow cavity megakaryocyte in pathological sections can be observed. Cell number and colony forming unit of megakaryocyte in vitro were significantly lower than those in wild type, and there was a significant difference between them (P0.05).
5. after the sublethal dose of TBI was given, the spleen index and thymus index of SRC-3~ (- / -) mice were slightly higher than that in the wild type, and there was no statistical difference compared with the wild type. It showed that the degree of post radiation injury in spleen and thymus of SRC-3~ (- / -) mice was lower than that of bone marrow injury, so the lymphatic group in SRC-3~ (- / -) mice The damage effect after irradiation is different from that of bone marrow.
6. after the sublethal dose of TBI was given, the level of IGF-1 in the serum of SRC-3~ (- / -) mice remained at a low level at each time point after illumination, and there was a statistical difference between the wild type and the wild type (P0.05). The serum hematopoietic factor IL-3 and IL-6 in the serum of.SRC-3~ (- / -) mice were significantly lower than the wild type at the seventh day after illumination, and there was a statistical difference between the two (P0.05) serum (P0.05). There was no difference in the level of cytokine TPO between the two groups.
7. the rate of apoptosis and the proportion of Sca-1+ cells contained in the BMNCs of the two groups of mice were measured by flow method. There was no significant difference between the apoptosis rate and the proportion of the Sca-1+ cells in the two mice of BMNCs. The apoptosis rate of BMNCs in SRC-3~ (- / -) mice after radiation was significantly increased and the proportion of Sca-1+ cells decreased significantly. The statistical difference (P0.05). At the same time, the expression of apoptotic protein p53 and Bax in BMNCs of SRC-3~ (- / -) mice was significantly increased after radiation, and the expression of NF- kappa B (p65) and Bcl-2 was significantly reduced in SRC-3~ (/ -) mice BMNCs, and there was a statistical difference between the apoptotic protein and the wild type (P0.05). The higher sensitivity of apoptosis is related to the abnormal expression of apoptosis related proteins.
8. the cell cycle and proliferation index of BMNCs in two groups of mice were measured by flow method. The cell cycle distribution and proliferation index of the two BMNCs mice were not statistically different in normal conditions. The BMNCs of SRC-3~ (- / -) mice after radiation showed a significant increase in the G0/G1 stage and the characteristics of the retardation of the S phase, and the proliferation index was also significantly lower than that in the wild. Type (P0.05). Further study found that the positive periodic regulators in the SRC-3~ (/ -) mouse BMNCs after radiation, including CyclinD1, CyclinE, CyclinA, CDK2, CDK4, were significantly reduced, and the protein expression of the negative cyclical regulator p53 and p21 increased significantly, and was statistically different from the wild type (P0.05). Moreover, SRC-3~ (- / -) mice The expression of p-AKT protein in s decreased after radiation, indicating that the SRC-3~ (- / -) mouse AKT proliferation signal pathway decreased after radiation. The above results suggest that BMNCs in SRC-3~ (/ -) mice has the characteristics of abnormal cell cycle distribution and proliferation disorder after radiation, in which the expression level of the cell and the active radiation of the AKT signaling pathway in the SRC-3~ (- / -) mice Reduction is an important reason for the slow recovery of hematopoiesis after radiation.
9. the proliferation activity of BMSCs in vitro cultured in vitro was measured by CKK-8. The proliferation activity of SRC-3~ (- / -) mice was significantly lower than that of the wild type (P0.05) in normal conditions, while the difference between the two groups was more significant (P0.01). And the CFU-F formation ability of SRC-3~ (- / -) mice was significantly lower than that in the wild type before and after illumination. There was a statistically significant difference (P0.05). In addition, we also detected the activity of AKT signaling pathway in the mouse BMSCs, and found that the p-AKT protein expression level in the two groups of mice was close to the level of the p-AKT protein expression in the BMSCs mice, but the p-AKT protein expression in the SRC-3~ (- / -) mice was significantly lower than that of the wild type in the SRC-3~ (- / -) mice after illumination, and there was a statistically significant difference (P0.05). The results showed that the proliferation ability and CFU-F formation ability of BMSCs in SRC-3~ (- / -) mice decreased before irradiation and increased after radiation, and the decrease of AKT proliferation pathway activity in SRC-3~ (/ -) mice after irradiation may be an important cause of the aggravation of the damage.
10. the level of cytokine in the culture supernatant of BMSCs cells in mice was measured by ELISA. Under normal conditions, the level of VCAM-1 and IGF-1 in the BMSCs supernatant of SRC-3~ (- / -) mice was lower than that in the wild type (P0.05). The average of VCAM-1, IGF-1, IL-3 and water in the BMSCs supernatant of SRC-3~ (- / -) mice was significantly lower after radiation injury. In wild type, there was a statistically significant difference between them (P0.05), indicating that SRC-3~ (- / -) mice had significantly lower BMSCs secreting cytokines than the wild type.
Conclusion:
1. the lack of SRC-3 can increase the mortality of mice after the lethal dose of TBI, indicating that SRC-3 can affect the survival rate of irradiated mice and improve the protective ability of mice to radiation injury on the whole level.
2. after the lack of SRC-3 can lead to sublethal dose of TBI, the damage of bone marrow in mice is heavier and the recovery of hematopoiesis is more slow. Therefore, SRC-3 can reduce the degree of hematopoietic injury after radiation in mice and promote the recovery of hematopoiesis, especially in megakaryocyte hematopoiesis.
3. SRC-3 is closely related to the maintenance of serum levels of IGF-1, IL-3 and IL-6. The lack of SRC-3 can lead to a significant decrease in the serum cytokine IGF-1 before and after irradiation, and the severe deficiency of the serum IL-3 and IL-6 levels in the serum after irradiation.
4. SRC-3 can protect the surviving Sca-1+ cells and the apoptotic cells in BMNCs after radiation, and reduce the apoptosis sensitivity of mice after BMNCs radiation by activating NF- kappa B (p65) protein and inhibiting the activity of p53 protein after radiation.
5. SRC-3 can promote the proliferation and cycle progression of BMNCs in mice after irradiation, reduce the proportion of G0/G1 phase, and reduce S phase arrest.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R818

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