本文選題：血管內皮生長因子 + 轉染��； 參考：《河北醫(yī)科大學》2015年博士論文

【摘要】：糖尿病外周血管病變(diabetic peripheral artery disease,PAD)是糖尿病(diabetic mellitus,DM)嚴重的慢性并發(fā)癥之一,與非DM患者相比,其主要病理改變?yōu)閯用}粥樣硬化,且多累及下肢遠端動脈,病變范圍更廣、呈多節(jié)段彌漫性的狹窄或閉塞,是導致DM足部壞疽、截肢的主要原因,嚴重影響DM患者的生存質量。目前傳統(tǒng)內科藥物、介入和手術治療等對遠端血管閉塞、流出道差的DM患者效果不佳,不能從根本上解決問題,并且此類DM患者多數(shù)高齡體弱,手術風險大,合并多種心腦血管病變,病情復雜,因此臨床治療上相當棘手,迫切需要尋求新的技術手段,如何促進血管新生實現(xiàn)血運重建成為治療關鍵。近年來干細胞移植成為發(fā)展迅速的一種全新治療模式,其中骨髓間充質干細胞(bone marrow mesenchymal stem cells,BMSCs)已被證實是一類具有多向分化潛能的干細胞,可以在一定的誘導條件下分化為骨、軟骨、脂肪、神經、心肌、血管內皮細胞等,參與不同組織的修復,但目前仍缺乏移植細胞在體內存活、分化、轉歸及參與血管再生的實驗依據(jù)可循,且骨髓MSCs采集風險較大,對患者年齡、身體條件、心理接受程度要求較高,在實際應用中,由于高糖、氧化應激、低氧等微環(huán)境使移植后MSCs的存活率非常低,新生血管形成速度慢等,極大地限制了干細胞移植治療的效果。相比之下,臍帶間充質干細胞(human umbilical cord mesenchymal stem cells,h UC-MSCs)與BMSCs在生物學特性方面極為相似,由于其來源更廣泛,采集方便,擴增力可塑性強,無免疫原性,不存在倫理學爭議,具有更加有效的MSCs潛能,有可能成為BMSCs的理想替代物,成為目前誘導分化最佳的種子細胞。對血循環(huán)的重建而言,目前已知有多種細胞因子和生長因子參與促進血管生成作用,其中血管內皮生長因子(Vascular endothelial growth factor,VEGF)通過與其血管內皮特異性受體結合,可顯著促進內皮增生及血管生成作用,被認為是機體內最強的促血管生長因子,但直接應用VEGF治療存在很多不足,如半衰期短、提純較為困難、應用量大、成本昂貴等,限制其臨床應用。因此,如何提高MSCs在缺血組織存活、分化,增加局部組織生長因子分泌量,促進新生血管形成,提高MSCs治療PAD療效是目前亟待解決的主要問題。本研究利用基因工程構建VEGF-EGFP基因表達載體,通過腺病毒轉染到h UC-MSCs細胞中,觀察基因轉染后對h UC-MSCs的生長增殖及目的基因VEGF表達情況;并利用增強型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)實現(xiàn)轉染后h UC-MSCs的活體示蹤定位;同時建立高脂喂養(yǎng)2型糖尿病SD大鼠下肢缺血模型,將轉染后的h UC-MSCs局部肌肉注射,觀察其向血管內皮分化促血管新生,側支循環(huán)的建立,改善下肢缺血的實驗療效;本研究應用h UC-MSCs作為VEGF基因治療平臺,不僅可通過提高VEGF持續(xù)分泌,達到局部穩(wěn)定的治療濃度,并通過VEGF的抗炎、抗氧化應激、抗凋亡、促進血管生成等作用改善缺血后微環(huán)境,為h UC-MSCs增殖、分化等過程提供最佳的生存空間;同時可放大h UC-MSCs的旁分泌作用,減少h UC-MSCs凋亡,提高h UC-MSCs移植后的定位歸巢和存活率等,有效發(fā)揮VEGF與h UC-MSCs在血管再生功效方面的協(xié)同倍增作用,從而為更好的發(fā)揮h UC-MSCs移植療效提供實驗基礎。本研究分為三部分:第一部分腺病毒介導VEGF轉染臍帶間充質干細胞的實驗研究目的:探討腺病毒載體介導VEGF轉染h UC-MSCs的可行性,以及VEGF轉染對h UC-MSCs形態(tài)及功能的影響。方法:構建VEGF-EGFP重組基因腺病毒載體,分離和培養(yǎng)h UC-MSCs,分為VEGF-EGFP轉染組、EGFP空載組及對照組,熒光倒置顯微鏡觀察細胞轉染效果,流式細胞儀測定細胞轉染效率,確定最佳病毒感染復數(shù)(multiplicities of infection,MOI);依據(jù)最佳MOI值轉染并收集細胞,應用蛋白印跡法(Western blot)、RT-PCR檢測轉染后目的基因VEGF的蛋白及m RNA表達情況;酶聯(lián)免疫吸附試驗(ELISA)檢測細胞培養(yǎng)上清VEGF蛋白水平,MTT法及流式細胞術評價基因轉染對h UC-MSCs增殖和細胞周期的影響。結果:熒光顯微鏡及流式細胞儀檢測提示腺病毒介導的VEGF-EGFP基因能夠成功轉染h UC-MSCs,且轉染效率高,對細胞結構形態(tài)無影響;目的基因VEGF在細胞內能夠轉錄和表達,并能分泌到細胞外,轉染后24h采用ELISA法在細胞培養(yǎng)上清中就檢測到有VEGF的表達和分泌,96h仍穩(wěn)定表達;與對照組和EGFP空載組相比,通過Western blot、RT-PCR檢測VEGF-EGFP轉染組VEGF表達水平升高顯著,且表達穩(wěn)定,可提高h UC-MSCs的抗凋亡及存活能力;MTT及流式細胞法檢測結果顯示VEGF基因轉染可提高h UC-MSCs增殖和分化能力。結論:腺病毒介導VEGF基因能夠成功轉染h UC-MSCs,能持續(xù)穩(wěn)定、高效表達VEGF,改善生存微循環(huán),提高h UC-MSCs的增殖及分化、存活能力,為開展VEGF基因轉染h UC-MSCs移植治療改善糖尿病下肢血管病變的可行性提供理論依據(jù)。第二部分高脂喂養(yǎng)2型糖尿病大鼠下肢缺血模型建立目的:目前普遍采用大鼠后肢股動脈結扎離斷的方法來制備后肢急性缺血模型,但對于如何建立及評估糖尿病高脂高血糖慢性動脈硬化閉塞癥的缺血狀態(tài),尚無一個穩(wěn)定有效慢性缺血模型及方法。方法:將大鼠20只隨機分為2組,DM組10只予以高脂喂養(yǎng)6個月,腹腔注射(Streptozocin,STZ)(35mg/kg)誘發(fā)糖尿病模型,對照組(10只)予以普食喂養(yǎng)6個月,成模DM組及對照組大鼠在麻醉后,消毒鋪巾,沿右下肢正中的皮膚縱行切開,于腹股溝下分離出股動脈,在腹股溝韌帶下切斷股動脈,近端結扎,隨后向遠端銳性剝離直至膝關節(jié),分離和結扎股動脈的所有分支,造成右下肢缺血模型,術后3d、7d、14d、28d觀察大鼠患肢活動狀況、肢體顏色、皮溫等,并于術后1d、3d、14d、28d常規(guī)麻醉后,保持溫度、光線相對恒定下,應用Peri Scan PIM3激光多普勒(Laser Doppler Perfusion Imaging,LDPI)行下肢血流監(jiān)測。術后28d麻醉動物后,切開后腹膜,于腹主動脈留置套管針,肝素鈉抗凝,以2 ml/s的速率注射造影劑約1.5 ml進行CT下肢血管造影。每組動物在血管造影后處死,分別取其健側和患側股四頭肌和腓腸肌行蘇木精-伊紅染色及CD31免疫組織化學染色、Western blot測定肌肉組織VEGF含量。結果:DM組有(8只)對照組(10只)制備成后肢缺血模型,術后第1d,兩組大鼠多普勒血流及CT血管造影均呈顯著降低提示缺血造模成功,兩組術后第7d和14d行激光多普勒提示缺血肢體血流有逐漸恢復趨勢,術后第28d DM組血流恢復較對照組顯著遲緩(P0.05);CT血管造影:DM組右下肢股動脈結扎處近端僅有少量血管代償性增加,遠心端仍無明顯血流;病理組織及免疫組化染色:術后第28d DM組缺血部位肌肉組織出現(xiàn)組織結構破壞,炎性細胞浸潤,毛細血管密度患側低于健側;缺血肌肉組織VEGF較對照組的蛋白表達明顯增加(P0.05)。結論:長期高脂喂養(yǎng)糖尿病模型基礎上,制作股動脈結扎離斷的下肢缺血模型,更接近于糖尿病下肢慢性缺血的情況,320排CT血管造影技術可以更立體直觀評估缺血狀態(tài),為進一步探討糖尿病下肢缺血病理機制及干細胞治療促血管新生等提供了較為理想的動物模型及評估指標。第三部分VEGF-EGFP轉染臍帶間充質干細胞移植治療改善糖尿病大鼠下肢缺血的實驗研究目的:通過腺病毒將重構的VEGF-EGFP基因轉染到h UC-MSCs細胞中,同時建立高脂喂養(yǎng)2型糖尿病SD大鼠下肢缺血模型,將轉染后h UC-MSCs局部下肢肌肉注射,觀察血管新生,側支循環(huán)建立,下肢缺血改善的實驗療效。方法:利用高脂飲食STZ誘導的2型糖尿病SD大鼠下肢股動脈結扎建立缺血模型后,將分組觀察VEGF-EGFP-h UC-MSCs、EGFP-h UC-MSCs、h UC-MSCs及PBS局部肌肉注射到下肢缺血部位,熒光倒置顯微鏡觀察移植細胞存活及定位;在治療后2、4周,激光多普勒(瑞典Peimed,LDPI)檢測局部血流;觀察下肢活動度和缺血情況;4周后利用腹主動脈結扎行下肢動脈CTA(東芝320層CT機)造影檢測雙下肢血管側支循環(huán)的形成;肌肉組織HE染色和CD31免疫組化檢測新生毛細血管數(shù)量及密度;RTPCR、Western blot等檢測組織標本中VEGF及MMP2,MMP9,TIMP1,TIMP2,ERK,AKt相關基因m RNA及蛋白的表達等。結果:移植后1、2、4周熒光顯微鏡下發(fā)現(xiàn),在下肢缺血部位有移植的EGFP標記的h UC-MSCs存活;移植后2、4周LDPI血流圖顯示VEGF-EGFP轉染組血流灌注的恢復水平要明顯高于EGFP空載組,移植4周后CT血管造影顯示VEGF-EGFP轉染組有新生側支循環(huán),HE及免疫組化染色顯示新生毛細血管明顯高均于空載組,RT-PCR及Western blot檢測組織標本VEGF-EGFP轉染組VEGF、ERK、AKt、MMP2和MMP9 m RNA和蛋白水平較其他兩組顯著增高,TIMP1、TIMP2的表達無明顯差異。結論:肌肉注射移植VEGF基因修飾后h UC-MSCs可在下肢缺血組織中定植存活,高效表達VEGF,促進內皮修復,比單純h UC-MSCs更能有效地促進血管新生,明顯改善糖尿病下肢缺血狀態(tài),為h UC-MSCs聯(lián)合基因治療糖尿病下肢血管病變提供新的理論依據(jù)。
[Abstract]:Diabetic peripheral vascular disease (diabetic peripheral artery disease, PAD) is one of the serious chronic complications of diabetes (diabetic mellitus, DM). Compared with non DM, the main pathological changes are atherosclerosis and many of the distal arteries of the lower extremity are involved in a wider range of lesions, with multiple segmental diffuse stenosis or occlusion, which leads to DM. Foot gangrene, the main cause of amputation, seriously affects the quality of life of DM patients. At present, the traditional medicine, intervention and surgical treatment are not effective for the distal vascular occlusion and the DM patients with poor outflow, and can not solve the problem fundamentally, and most of these DM patients are weak in age, the operation risk is large, and many kinds of cardiovascular and cerebrovascular diseases are merged. The clinical treatment is very difficult, so it is urgent to seek new techniques. How to promote blood vessel revascularization is the key to the treatment. In recent years, stem cell transplantation has become a rapid development of a new treatment model, in which bone marrow mesenchymal stem cells (BMSCs) has been proved to be A class of stem cells with multiple differentiation potential can differentiate into bone, cartilage, fat, nerve, myocardium, vascular endothelial cells and so on under certain induction conditions, and participate in the repair of different tissues. However, there is still a lack of experimental basis for the survival of the transplanted cells in the body, differentiation, transformation and involvement of vascular regeneration, and the risk of collecting bone marrow MSCs is more than that of the bone marrow. In practical application, the survival rate of MSCs after transplantation is very low and the rate of angiogenesis is slow, which greatly restricts the effect of stem cell transplantation. In contrast, umbilical cord mesenchymal stem cells (human umbilical). Cord mesenchymal stem cells, H UC-MSCs) and BMSCs are very similar in biological characteristics. Because of their more extensive origin, convenient collection, strong extenability, no immunogenicity, no ethical controversy, more effective MSCs potential, may become an ideal substitute for BMSCs and become the best seed to induce differentiation at present. For the reconstruction of blood circulation, a variety of cytokines and growth factors are known to be involved in promoting angiogenesis, in which Vascular endothelial growth factor (VEGF) can significantly promote endothelial proliferation and angiogenesis by combining with its vascular endothelial specific receptor. It is considered to be within the body. The strongest angiogenic growth factor, but the direct application of VEGF has many shortcomings, such as short half-life, more difficult purification, large amount of application, high cost and so on, which restrict its clinical application. Therefore, how to improve the survival and differentiation of MSCs in the ischemic tissue, increase the secretion of local tissue growth factor, promote the formation of new blood vessels and improve the treatment of MSCs for the treatment of PAD The effect is the main problem to be solved at present. This study uses gene engineering to construct VEGF-EGFP gene expression vector and transfect the adenovirus into H UC-MSCs cells, observe the growth and proliferation of H UC-MSCs and the expression of the target gene VEGF after gene transfection; and use the enhanced green color fluorescent protein (enhanced green fluorescent protein,) EGFP) in vivo tracer localization of H UC-MSCs after transfection; at the same time, a high fat feeding type 2 diabetic SD rat lower limb ischemia model was established, and the transfected h UC-MSCs was injected locally to observe its angiogenesis to vascular endothelium, the establishment of collateral circulation, and the improvement of the experimental efficacy of lower extremity blood deficiency. This study used h UC-MSCs as a VEGF base. Because of the treatment platform, it can not only improve the concentration of local stable treatment by increasing the continuous secretion of VEGF, but also improve the microenvironment after ischemia by the anti-inflammatory, antioxidant stress, anti apoptosis, and angiogenesis of VEGF, and provide the best living space for H UC-MSCs proliferation, differentiation and other processes. At the same time, the paracrine effect of H UC-MSCs can be amplified and reduced. Less h UC-MSCs apoptosis, improve the localization and survival rate after H UC-MSCs transplantation, effectively play a synergistic multiplier effect of VEGF and H UC-MSCs in vascular regeneration, thus providing an experimental basis for the better efficacy of H UC-MSCs transplantation. This study is divided into three parts: the first part of adenovirus mediated VEGF transfection of umbilical cord mesenchymal stem cells Objective: To investigate the feasibility of adenovirus vector mediated VEGF transfection of H UC-MSCs and the effect of VEGF transfection on the morphology and function of H UC-MSCs. Methods: construct VEGF-EGFP recombinant adenovirus vector, isolate and culture h UC-MSCs, divide into VEGF-EGFP transfection group, EGFP empty load group and control group, and observe cell transformation by fluorescence inverted microscope. The transfection efficiency was determined by flow cytometry, the optimal number of multiplicities of infection (MOI) was determined, and the cells were transfected and collected according to the optimum MOI value. The expression of the egg Rhizoma Bletillae m RNA expression of the target gene VEGF after transfection was detected by the Western blot (Western blot), and the enzyme linked immunosorbent assay (ELISA) was finely detected. Cell culture supernatant VEGF protein level, MTT method and flow cytometry to evaluate the effect of gene transfection on the proliferation and cell cycle of H UC-MSCs. Results: fluorescence microscopy and flow cytometry showed that adenovirus mediated VEGF-EGFP gene could transfect h UC-MSCs successfully, and the transfection efficiency was high, and the cell structure morphology was not affected; the target gene VEGF was found. The cells can be transcribed and expressed, and can be secreted out of the cell. After transfection, the expression and secretion of VEGF are detected by ELISA method in cell culture supernatant, and the expression of 96h is still stable. Compared with the control group and the EGFP empty load group, the expression level of VEGF in the VEGF-EGFP transfer group is increased by Western blot and RT-PCR, and the expression is stable, and the expression is stable and can be extracted. The expression of 24h is stable and can be extracted. The anti apoptosis and survival ability of Gao H UC-MSCs, and the results of MTT and flow cytometry showed that VEGF gene transfection could improve the proliferation and differentiation of H UC-MSCs. Conclusion: adenovirus mediated VEGF gene can successfully transfect h UC-MSCs, can continue to be stable, efficiently express VEGF, improve the survival microcirculation, improve the proliferation and differentiation of H UC-MSCs, and the viability, To provide a theoretical basis for the feasibility of VEGF gene transfection of H UC-MSCs transplantation in the treatment of diabetic lower extremity vascular lesions. Second the purpose of establishing the lower limb ischemia model of type 2 diabetic rats is to establish an acute ischemia model of the hind limbs by the method of ligature of the femoral artery in the hind limbs of the rat. And to assess the ischemic state of chronic arteriosclerosis obliterans with hyperglycemia and hyperglycemia, there was no stable and effective chronic ischemia model and method. Methods: 20 rats were randomly divided into 2 groups, 10 in group DM were fed with high fat for 6 months, and the diabetes model was induced by intraperitoneal injection (Streptozocin, STZ) (35mg/ kg), and the control group (10) was fed by common food (6). After anaesthesia, rats in the model DM group and the control group sterilize the tissue and cut the skin along the right lower extremities, separate the femoral artery under the groin, cut the femoral artery under the groin, and ligate the femoral artery near the inguinal ligament. Then the distal end is stripped to the knee, and all branches of the femoral artery are separated and ligated to cause ischemic model of the right lower limb. After the operation, 3D, 7d, 14d, 28d were used to observe the condition of limb movement, body color, skin temperature and so on. After the routine anesthesia of 1D, 3D, 14d, 28d, the temperature was maintained. The light ray was relatively constant, and the Peri Scan PIM3 laser Doppler was used to monitor the blood flow of the lower limbs. After the operation, the peritoneum was opened and the peritoneum was opened and the abdominal initiative was taken after the operation. Vein indwelling trocar, heparin sodium anticoagulant, CT lower extremity angiography was performed by injection of contrast agent about 1.5 mL at 2 ml/s rate. Each group of animals died after angiography. The healthy side and the affected lateral femoral four head and the gastrocnemius muscle were stained with hematoxylin eosin staining and CD31 immunohistochemical staining, and Western blot was used to determine the VEGF content in muscle tissue. Results: DM Group (8) control group (10 rats) was prepared to form a hind limb ischemia model. After operation 1D, the Doppler blood flow and CT angiography in the two groups were significantly reduced to suggest that the ischemia model was successful. The two groups of 7D and 14d after the operation showed that the blood flow of the ischemic limb was gradually restored, and the blood flow recovery of group 28d DM after operation was significantly slower than that of the control group (P0 .05); CT angiography: in group DM, only a small amount of blood vessels were compensated for the proximal femoral artery ligation at the right lower extremities, and there was no obvious blood flow in the distal end of the heart. Pathological tissue and immunohistochemical staining: tissue structure destruction, inflammatory cell infiltration, capillary density side lower than the healthy side, and VEGF of ischemic muscle tissue in group 28d DM after operation. The expression of protein in the control group was significantly increased (P0.05). Conclusion: on the basis of long-term high fat feeding diabetes model, the ischemia model of lower extremity of the femoral artery ligation is closer to the condition of chronic ischemia in the lower limbs of diabetes. The 320 row CT angiography can be more stereoscopic to assess the ischemic state more stereoscopic, to further explore the deficiency of the lower extremity of diabetes. Blood pathological mechanism and stem cell therapy provide an ideal animal model and evaluation index. Third VEGF-EGFP transfection of umbilical cord mesenchyme stem cell transplantation to improve lower limb ischemia in diabetic rats: transfection of recombinant VEGF-EGFP gene into H UC-MSCs cells by adenovirus. To establish the lower limb ischemia model of SD rats with type 2 diabetes mellitus by high fat feeding, the local muscle injection of H UC-MSCs after transfection was injected to observe the effects of angiogenesis, collateral circulation and lower limb ischemia improvement. Methods: after the ischemia model was established by ligation of the femoral artery in the lower extremities of type 2 diabetic SD rats induced by high fat diet STZ, the group was divided into groups to observe VEGF-EG. FP-h UC-MSCs, EGFP-h UC-MSCs, H UC-MSCs and PBS were injected into the ischemic parts of the lower extremities. The survival and location of the transplanted cells were observed by fluorescence inverted microscope. The local blood flow was detected by laser Doppler (Sweden Peimed, LDPI) after the treatment. The lower extremity activity and the blood deficiency were observed. 4 weeks later, the lower extremity artery was ligated by abdominal aorta ligature. The formation of collateral circulation of both lower extremity vessels was detected by Toshiba 320 layer CT. The number and density of newborn capillaries were detected by HE staining and CD31 immunohistochemistry in muscle tissue; VEGF and MMP2, MMP9, TIMP1, TIMP2, ERK, AKt related genes and protein expression in tissue specimens, such as RTPCR, Western blot, etc. The EGFP labeled h UC-MSCs transplanted in the lower limb of the lower extremities was found to survive, and the LDPI blood flow map of 2,4 weeks after transplantation showed that the recovery level of the blood flow perfusion in the VEGF-EGFP transfected group was significantly higher than that of the EGFP no-load group. After 4 weeks, the CT angiography showed that there was a new collateral circulation in the VEGF-EGFP transfection group, and HE and immunohistochemical staining showed the newborn capillary. RT-PCR and Western blot detected VEGF-EGFP transfection group VEGF, ERK, AKt, MMP2 and MMP9 m significantly higher than the other two groups. VEGF, which promotes endothelial repair, is more effective than h UC-MSCs in promoting angiogenesis, obviously improving the state of lower limb ischemia in diabetes, and providing a new theoretical basis for the treatment of diabetic lower extremity vascular lesions by H UC-MSCs combined gene.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R587.2

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2 徐祗順,蔣曉剛,劉海南,傅強,許復郁,劉玉強;1型糖尿病大鼠陰莖神經型一氧化氮合酶的含量及意義[J];山東醫(yī)科大學學報;2001年04期

3 艾智華,孟萍,蔡紅衛(wèi);糖尿病大鼠血、尿、腎臟內皮素變化及意義[J];第三軍醫(yī)大學學報;2002年08期

4 李全宏,田澤,蔡同一;南瓜提取物對糖尿病大鼠降糖效果研究[J];營養(yǎng)學報;2003年01期

5 張斌,高鑫;絲裂原激活蛋白激酶信號通路與糖尿病并發(fā)癥[J];國外醫(yī)學.內分泌學分冊;2004年05期

6 丁文成;;運動在糖尿病治療中的作用機理及實施原則[J];貴州體育科技;2004年04期

7 梁俊清,丁春華,凌亦凌;糖尿病時肺內氧化與抗氧化系統(tǒng)失衡的研究進展[J];河北醫(yī)科大學學報;2005年01期

8 陳磊,章新華,聶宏光,金萬寶,李金鳴;早期糖尿病大鼠血中降鈣素基因相關肽的變化[J];中國醫(yī)科大學學報;2005年01期

9 李紅,張哲,翁紅雷,阮昱;糖尿病大鼠腎臟α平滑肌肌動蛋白的表達[J];浙江大學學報(醫(yī)學版);2005年02期

10 李潔;趙明;王燕燕;;糖尿病足的分子生物學機制[J];中國臨床康復;2006年12期

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1 王志強;陳秀榮;武兵;;(綜述)中藥治療糖尿病及其并發(fā)癥的臨床科研進展[A];中國中醫(yī)藥學會基層中醫(yī)藥會議�？痆C];1997年

2 金滿文;韓毅;王妍;陳雷;何婷;沈紀中;辛欣;李先輝;胡燕;;五甲基槲皮素全面防治2型糖尿病的作用及機制[A];中國藥理學會第十一次全國學術會議�？痆C];2011年

3 周水平;仝小林;;糖尿病陽痿的研究進展[A];糖尿病中醫(yī)研究進展——全國第六次中醫(yī)糖尿病學術會議論文集[C];2000年

4 黃慧;田浩明;李雄偉;;殼聚糖胰島素微球在糖尿病大鼠中的降糖作用研究[A];中華醫(yī)學會第六次全國內分泌學術會議論文匯編[C];2001年

5 柳剛;關廣聚;亓同鋼;傅余芹;李學剛;孫云;吳濤;文蓉珠;;丹參對糖尿病大鼠腎臟的保護作用及其機制[A];第六次全國中西醫(yī)結合血瘀證及活血化瘀研究學術大會論文匯編[C];2005年

6 ;血管緊張素-(1-7)對糖尿病大鼠腎臟影響的研究[A];2005年浙江省內科學學術年會論文匯編[C];2005年

7 仝小林;劉銅華;陳良;;中醫(yī)藥防治糖尿病及其并發(fā)癥研究20年概況及展望[A];第九次全國中醫(yī)糖尿病學術大會論文匯編[C];2006年

8 祁少海;劉坡;舒斌;謝舉臨;徐盈斌;黃勇;毛任翔;劉旭盛;;不同深度糖尿病大鼠燙傷模型的制備[A];第五屆全國燒傷救治專題研討會燒傷后臟器損害的臨床救治論文匯編[C];2007年

9 韓亭亭;蘇布德格日樂;胡耀敏;劉偉;;2型糖尿病大鼠在急性炎癥狀態(tài)下的反應能力研究[A];中華醫(yī)學會第十次全國內分泌學學術會議論文匯編[C];2011年

10 陳向芳;劉志民;石勇銓;鄒俊杰;湯瑋;馮曉云;張貝;張?zhí)m予;陽秋良;許娟;岳欣欣;;糖尿病大鼠“內源性損害”的作用機制[A];中華醫(yī)學會第十次全國內分泌學學術會議論文匯編[C];2011年

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1 郭賽珊　梁曉春 潘明政;中西醫(yī)結合治療糖尿病慢性并發(fā)癥可顯著改善癥狀[N];中國中醫(yī)藥報;2007年

2 本報記者 王樂羊;中西醫(yī)結合防治糖尿病大有可為[N];中國中醫(yī)藥報;2002年

3 北京協(xié)和醫(yī)院 梁曉春;對中醫(yī)治糖尿病并發(fā)癥研究的思考[N];中國中醫(yī)藥報;2011年

4 劉燕玲;肥胖是糖尿病的源頭[N];健康報;2006年

5 仝小林;糖尿病慢性并發(fā)癥論治[N];中國中醫(yī)藥報;2003年

6 汪敏;糖尿病皮膚易損元兇找到[N];衛(wèi)生與生活報;2003年

7 林蘭;中西醫(yī)結合治療糖尿病的前景[N];中國中醫(yī)藥報;2007年

8 北京協(xié)和醫(yī)院中醫(yī)科主任 梁曉春;糖尿病周圍神經病變的中西醫(yī)治療進展[N];中國醫(yī)藥報;2009年

9 本報記者 劉艷芳;糖尿病干預不能忽視抗氧化[N];中國食品報;2012年

10 譚小月;糖尿病與腎病關系研究的新進展[N];中國中醫(yī)藥報;2004年

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1 毛雪琴;富釩鷹嘴豆芽對糖尿病大鼠糖脂代謝及學習記憶的影響[D];山東大學;2008年

2 羅禮達;溫臟扶正驅邪法對2型糖尿病T淋巴細胞免疫功能影響的臨床研究[D];廣州中醫(yī)藥大學;2009年

3 高泓;參芪復方調控糖尿病血管PI3-K/Akt通路的實驗研究[D];成都中醫(yī)藥大學;2009年

4 張冬梅;特發(fā)性1型糖尿病的臨床研究[D];中南大學;2003年

5 王寬宇;早期離斷配合外用中藥藥膜治療糖尿病足的實驗研究和臨床觀察[D];黑龍江中醫(yī)藥大學;2010年

6 李純;抑制神經酰胺合成對糖尿病大鼠內皮功能紊亂及動脈粥樣硬化形成的影響[D];中南大學;2011年

7 郭志新;血管緊張素Ⅱ受體拮抗劑對糖尿病大鼠腎臟的保護作用及其作用機制的研究[D];天津醫(yī)科大學;2002年

8 宋莉莉;糖尿病對血管性癡呆認知功能影響的實驗研究[D];第二軍醫(yī)大學;2004年

9 姜兆順;基于結構化住院病歷采集系統(tǒng)對糖尿病及血管并發(fā)癥辨證論治規(guī)律的研究[D];中國中醫(yī)研究院;2005年

10 郝賢;降糖消脂湯對2型糖尿病大鼠胰島素抵抗影響機制實驗研究[D];黑龍江中醫(yī)藥大學;2006年

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1 高峰;骨髓間充質干細胞不同移植方法治療糖尿病的實驗研究[D];暨南大學;2009年

2 吳迪;電針治療糖尿病周圍神經病的臨床觀察及其作用機制的實驗研究[D];黑龍江中醫(yī)藥大學;2009年

3 張紅霞;2型糖尿病患者血清tHcy水平與認知功能障礙的關系[D];山東大學;2009年

4 侯亞利;2型糖尿病患者心臟結構和功能的變化[D];蘭州大學;2010年

5 袁海潑;通絡糖泰方對糖尿病周圍神經病變大鼠血清IL-6水平表達影響的研究[D];成都中醫(yī)藥大學;2009年

6 張麗;2型糖尿病患者的肺功能變化及其相關因素分析[D];新疆醫(yī)科大學;2010年

7 程晶;半導體激光聯(lián)合高壓電位對糖尿病大鼠血管病變作用機理研究[D];黑龍江中醫(yī)藥大學;2010年

8 邱作成;健脾腎化瘀濁法治療糖尿病多發(fā)性周圍神經病變的研究[D];新疆醫(yī)科大學;2004年

9 馮智敏;糖尿病對大鼠正畸牙齒移動的影響[D];河北醫(yī)科大學;2006年

10 鄭淑君;胰島素樣生長因子-1與1型糖尿病大鼠骨骼肌病變的關系[D];山西醫(yī)科大學;2006年

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