大鼠神经干细胞中PI3-K/AKT信号转导通路的研究

目的 应用PI3-K特异性阻滞剂LY294002孵育大鼠神经干细胞(NSCs),探讨PI3-K/AKT信号转导通路在NSCs中的作用.方法 体外分离、培养E15～16 胎鼠NSCs,应用不同浓度(0~40 μmol/L)的LY294002孵育NSCs后进行细胞存活(WST-8检测)、增殖(BrdU免疫组织化学鉴定)、分化(β Tubulin-Ⅲ免疫组织化学鉴定)和AKT磷酸化蛋白水平(Western Blotting)的检测.结果 LY294002对NSCs的存活、增殖和分化的影响呈剂量依赖性.在LY294002较高浓度(25、30、35、40 μmol/L)时,NSCs的存活率明显下降(P<0.05);LY294002为30、35、40 μmol/L时,BrdU阳性细胞数明显少于正常对照组(LY294002 0 μmol/L)(P<0.05);LY294002为35、40 μmol/L时,β Tubulin-Ⅲ的阳性细胞数明显少于正常对照组(P<0.05).LY294002 阻断AKT表达的作用呈剂量依赖性.结论 PI3-K/AKT信号转导通路对大鼠NSCs的存活、增殖、分化起重要作用.     

肠三叶因子对胃黏膜上皮细胞增殖的影响及其信号转导机制的研究

目的：肠三叶因子（ intestinal trefoil factor , ITF）对胃黏膜具有保护作用,但其机制尚不明确。文中探讨重组ITF对胃黏膜上皮细胞增殖能力的影响及其可能作用的分子机制。方法将体外培养的GES-1细胞分为3组,其中1组作为细胞增殖活力实验空白对照组,其他2组分别加入浓度为100和500 ng/mL的人重组ITF形成低浓度ITF组和高浓度ITF组。光镜下观察细胞形态,并采用CCK-8试剂盒检测GES-1细胞的增殖活力。体外培养的GES-1细胞株分别用100 ng/mL的ITF和浓度为15μmol/L的PI3 K/Akt 信号通路抑制剂LY294002进行处理,分为信号通路蛋白表达实验空白对照组、ITF组、LY294002组和ITF＋LY294002组,光镜下观察细胞形态。随后采用CCK-8试剂盒检测GES-1细胞处理后的增殖活力,采用Western blot检测PI3K/Akt信号通路中p-Akt和Akt蛋白的表达情况。结果与空白对照组相比, ITF刺激下GES-1细胞增殖活力明显增强,且ITF浓度越高、增殖活力越强。使用LY294002处理后,能够显著抑制ITF刺激的细胞增殖。 Western blot检测结果表明,ITF提高了p-Akt蛋白的表达水平,激活PI3K/Akt信号通路。结论 ITF促进GES-1细胞的增殖,推测其分子作用机制主要是通过激活PI3K/Akt信号通路促进细胞增殖。     

大鼠神经干细胞中PI3-K/AKT信号转导通路的研究

目的 应用PI3-K特异性阻滞剂LY294002孵育大鼠神经干细胞（NSCs）,探讨PI3-K/AKT信号转导通路在NSCs中的作用。方法 体外分离、培养E15～16 胎鼠NSCs,应用不同浓度（0~40 μmol/L）的LY294002孵育NSCs后进行细胞存活(WST-8检测)、增殖(BrdU免疫组织化学鉴定)、分化(β Tubulin-Ⅲ免疫组织化学鉴定)和AKT磷酸化蛋白水平(Western Blotting)的检测。结果 LY294002对NSCs的存活、增殖和分化的影响呈剂量依赖性。在LY294002较高浓度（25、30、35、40 μmol/L）时,NSCs的存活率明显下降（P<0.05）;LY294002为30、35、40 μmol/L时,BrdU阳性细胞数明显少于正常对照组（LY294002 0 μmol/L）（P<0.05）;LY294002为35、40 μmol/L时,β Tubulin-Ⅲ的阳性细胞数明显少于正常对照组（P<0.05）。LY294002 阻断AKT表达的作用呈剂量依赖性。结论 PI3-K/AKT信号转导通路对大鼠NSCs的存活、增殖、分化起重要作用。     

Ghrelin对脂肪间充质干细胞神经分化的影响

目的 探讨Ghrelin联合LY294002对脂肪间充质干细胞（ADSCs）神经分化的影响,并阐明其作用机制。 方法 倒置相差显微镜下观察ADSCs形态表现,流式细胞术鉴定ADSCs表面抗体阳性表达率,将第4代ADSCs分为对照组（不进行干预）、LY294002组［给予40 μmol·L^－1磷酸酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）通路特异抑制剂LY294002］、Ghrelin组（给予0.1 μmol·L^－1 Ghrelin）、Ghrelin+LY294002组（给予40 μmol·L^－1 LY294002+0.1 μmol·L^－1 Ghrelin）。倒置相差显微镜下观察各组ADSCs神经分化后的形态表现,免疫荧光染色法检测各组ADSCs中神经元特异性烯醇化酶（NSE）、巢蛋白（Nestin）和胶质纤维酸性蛋白（GFAP）阳性细胞百分率,Western blotting法检测各组ADSCs中 PI3K/Akt通路蛋白表达水平,并计算磷酸化PI3K（p-PI3K）/PI3K和磷酸化Akt（p-Akt）/Akt比值。 结果 ADSCs表面抗体CD90和CD13高表达,CD34、CD45和CD106低表达。与对照组比较,LY294002组仅部分细胞由长梭形转变为类圆形胞体,突起较短、数量减少,而Ghrelin组细胞由长梭形转变为类圆形胞体速度增快;与LY294002组比较,Ghrelin+ LY294002组细胞由长梭形转变为类圆形胞体速度增快,且突起数量增加。与对照组比较,LY294002组ADSCs中NSE和Nestin阳性细胞百分率及p-PI3K/PI3K和p-Akt/Akt比值明显降低（P<0.05）,Ghrelin组ADSCs中NSE和Nestin阳性细胞百分率及p-PI3K/PI3K和p-Akt/Akt比值升高（P<0.05）;与LY294002组比较,Ghrelin+LY294002组ADSCs中NSE和Nestin阳性细胞百分率及p-PI3K/PI3K和p-Akt/Akt比值升高（P<0.05）;各组ADSCs中GFAP阳性细胞百分率比较差异无统计学意义（P>0.05）。 结论 Ghrelin通过激活PI3K/Akt通路促进ADSCs神经分化。     

胰岛素样生长因子-2对婴幼儿血管瘤干细胞增殖及脂肪分化的影响

目的 探讨胰岛素样生长因子-2( IGF-2)对婴幼儿血管瘤干细胞(HemSCs)增殖及向脂肪分化的影响.方法 搜集3例增生期草莓状婴幼儿血管瘤术后组织,采用胶原酶消化分离后CD133免疫磁珠吸附得到血管瘤干细胞. CCK-8法检测不同浓度IGF-2对HemSCs增殖的影响,Western blot检测空白对照组及 IGF-2 组(100 ng/ml)、IGF-2 +OSI-906 组(IGF-2: 100 ng/ml; OSI-906: IGF-1R受体抑制剂1 μmol/L)、IGF-2+LY294002 组(LY294002: PI3K 抑制剂10 μmol/L)、LY294002组( 10 μmol/L)各组细胞中脂肪转化因子 C/EBPα、C/EBPβ、PPARγ、adiponectin、p-AKT及total AKT的表达.结果 IGF-2在体外对HemSCs增殖有明显促进作用( P＜0. 05);Western blot法显示,与 IGF-2 +OSI-906 组、IGF-2+LY294002 组、 LY294002 组及空白对照组比较, IGF-2 组C/EBPα、C/EBPβ、 PPARγ、 adiponectin 表达量增加( P ＜0. 05);与IGF-2+OSI-906组、IGF-2+LY294002组及空白对照组比较, IGF-2 组 p-AKT 表达量增加( P ＜0. 05).结论 IGF-2可能通过 IGF-1R/PI3K/AKT通路影响 HemSCs的脂肪转化过程.     

LY294002对人纤维肉瘤HT1080细胞生长的影响及机制研究

目的 研究PI3K特异性抑制剂LY294002对人纤维肉瘤HT1080细胞生长的影响及机制.方法 将对数生长期的HT1080细胞分组培养,对照组加入不含LY294002的培养液,实验组加入LY294002,浓度分别为5、10、25、50、100 μmol/L,培养12 h和24 h后,用SunBio~(TM) Am-Blue法测定细胞增殖抑制率.100 μmol/L LY294002作用HT1080细胞24 h后,观察细胞形态的改变,用流式细胞仪检测细胞凋亡情况,并收集蛋白标本,Western Blotting检测p-Akt及p-mTOR蛋白的表达.结果 与对照组比较,随着LY294002剂量、作用时间的增加,对HT1080细胞增殖抑制率增大(P＜0.05).LY294002(100 μmol/L)作用24 h后HT1080细胞胞体皱缩,细胞数较对照组减少,细胞早期凋亡率较对照组增加(P＜0.05),p-Akt(0.23±0.01)及p-mTOR(0.32±0.06)蛋白的表达低于对照组p-Akt(0.63±0.02)及p-mTOR(0.71±0.02)蛋白的表达(P＜0.01).结论 LY294002通过抑制PI3K-mTOR信号通路来抑制HT1080细胞的生长,PI3K-mTOR信号通路参与纤维肉瘤细胞的生长调节,该通路可能作为治疗纤维肉瘤的新靶点.     

苦参碱联合LY294002对人髓系白血病K562细胞增殖、凋亡和细胞周期的影响

目的 探究苦参碱（Mat）联合LY294002对人髓系白血病K562细胞增殖、凋亡和细胞周期的影响及可能机制。方法 采用CCK-8法检测不同浓度Mat单药和联合LY294002对K562细胞增殖的影响;将K562细胞分为对照组、Mat组、LY294002组和Mat+LY294002组,分别采用0.4 g/L Mat及10 μmol/L LY294002单独或联合干预48 h后,应用光学显微镜观察细胞形态变化,流式细胞术annexin Ⅴ-FITC/PI双标记检测细胞凋亡,PI单标记检测细胞周期变化,Western blot法检测Mat联合LY294002对K562细胞p-mTOR、p-PI3K、Akt、p-Akt、CyclinD1、Bcl-2、Caspase-9蛋白表达的影响。结果 CCK-8结果显示,不同浓度Mat单药和联合LY294002均可抑制K562细胞的增殖,具有时间和浓度依赖性,且联合组增殖抑制率比单药组明显升高（P<0.01）。形态学检查显示,联合用药组比单药组凋亡表现更明显。流式细胞结果显示,与对照组及单药组相比,联合组显著促进细胞凋亡（[ 42.50±2.63）%,P<0.01],显著增加了细胞周期G0/G1期细胞比率（[ 57.23±1.44）%,P<0.01]。Western blot结果表明,两药联合后K562细胞p-mTOR、CyclinD1、p-PI3K、p-Akt、Bcl-2蛋白表达水平明显增高（P<0.01）,Caspase-9蛋白表达水平下降（P<0.01）。结论 Mat和LY294002联合应用可以增强对K562细胞生长抑制的协同作用,其机制可能是通过有效下调PI3K/Akt信号通路中的p-Akt表达,使p-mTOR、CyclinD1和Bcl-2蛋白表达下调,Caspase-9蛋白表达上调来实现的。     

LY294002联合Znpp IX对人乳腺癌细胞增殖及侵袭能力的影响

[目的]探讨PI3K/Akt信号通路抑制剂LY294002联合血红素加氧酶-1(HO-1)抑制剂Znpp IX对人乳腺癌MCF7/Adr细胞增殖及侵袭能力的影响.[方法]将Znpp IX(0.625~10.000μmol/L)与LY294002(5~80μmol/L)单独或联合应用于人乳腺癌MCF7/Adr细胞,用MTT法检测药物对MCF7/Adr细胞增殖的影响.实验分为对照组、LY294002组(10μmol/L)、Znpp IX组(1.250μmol/L)和联合用药组(LY294002 10μmol/L+Znpp IX1.250μmol/L),用Transwell小室测定各组MCF7/Adr细胞的侵袭力和迁移力,Western blot法检测Akt,p-Akt和HO-1蛋白的表达水平.[结果]联合用药组、LY294002组及Znpp IX组MCF7/Adr细胞的增殖抑制率分别为58.60%±3.24%,42.50%±2.44%和39.70%±3.21%,联合用药组MCF7/Adr细胞的增殖抑制率明显高于LY294002组和Znpp IX组(P<0.05).LY294002和Znpp IX单独或联合应用均可显著抑制人乳腺癌MCF7/Adr细胞的侵袭和转移(P<0.05),其中联合用药组抑制作用显著优于LY294002组和Znpp IX组(P<0.05).Western blot检测结果显示,各组Akt蛋白表达水平间差异无统计学意义(P>0.05);LY294002组p-Akt,HO-1表达水平均明显低于对照组(P<0.05),Znpp IX组HO-1表达水平明显低于对照组(P<0.05),与LY294002组和Znpp IX组比较,联合用药组p-Akt,HO-1水平均明显降低(P<0.05).[结论]Znpp IX和LY294002单独应用均可抑制人乳腺癌MCF7/Adr细胞的增殖、侵袭及转移,联合应用时作用加强,提示Znpp IX和LY294002联合应用具有协同增效作用.     

CTGF诱导的人胚肺成纤维细胞转分化中PI3K/Akt与p38MAPK磷酸化的关系

目的 探讨结缔组织生长因子(CTGF)诱导的人胚肺成纤维细胞(HFL-I)转分化中磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)和丝裂原活化蛋白激酶(p38MAPK)通路的活动状态以及相关性的研究.方法 将体外培养的HFL-I分成4组:(1)CTGF处理组(20 ng/ml);(2) LY294002(10 μmol/L)预处理60min,再行CTGF刺激(20 ng/ml):(3)SB203580 (10 μmol/L)预处理60 min,再行CTGF刺激(20 ng/ml); (4)LY294002(10 μmol/L)和SB203580(10 μmol/L)联合作用60 min,再加入CTGF(20 ng/ml).选取不同时相点,Western blot测定平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、p-Akt和Akt的蛋白表达:RT-PCR检测PI3K和p38MAPKmRNA表达.结果 与对照组相比,CTGF诱导15 min后p-Akt水平升高,30 min时达至顶峰(P＜0.01).CTGF诱导24h后α-SMA表达显著升高(P＜0.01),可被LY294002阻断.LY294002和SB203580单独或联合预处理后,显著抑制CTGF诱导的p-Akt活化(P＜0.01).结论 CTGF诱导的成纤维细胞-肌成纤维细胞转分化中有PI3K/Akt通路的激活,PI3K和p38MAPK均可以调节Akt的活性,其特异性抑制剂均可阻断Akt的磷酸化.     

PI3K/AKT信号途径在大鼠脑源性神经干细胞中的作用

目的 探讨磷脂酰肌醇-3激酶（phosphatidylinositol-3 kinase,PI3K）/AKT信号途径在体外培养神经干细胞存活、分化中的作用.方法 分为对照组、PI3K特异性抑制剂LY294002组（LY294002浓度分别为2 μmol/L,5 μmol/L,10 μmol/L）;采用间接免疫荧光法检测Nestin、NF-200和GFAP的表达;TUNEL评价细胞凋亡率;Western Blot法检测磷酸化的AKT的表达.结果 随着LY294002浓度增大,神经干细胞的形态变化逐渐明显,而且细胞凋亡率也逐渐增加.低浓度时（2 μmol/L）神经干细胞凋亡率与对照组无显著性差异（P〉0.05）;高浓度时（5 μmol/L、10 μmol/L）神经干细胞凋亡率与对照组、低浓度组有极显著性差异（P〈0.01）.Western Blot结果提示,随着LY294002浓度增大,磷酸化AKT的表达逐渐减弱.结论 本研究提示神经干细胞存活依赖于PI3K/AKT途径的活化,其机制可能是PI3K/AKT活化后,抑制了细胞凋亡事件的发生,但PI3K/AKT途径可能在神经干细胞分化中的作用甚微.     

Acupuncture Treatment of Vomiting

CASE HISTORY A female patient, 46 years old, head of the foreign affairs department of a certain university in Beijing, paid her first visit on October 9, 2006, with the chief complaint of vomiting for one month. She got vomiting after meals in early September. Before that, she had discomfortable sensation in the stomach due to angry with others, but she didn't pay much attention. Later, it developed into vomiting after eating. After the vomiting, the discomfort would be relieved, but with slight hypodynamia. She was once diagnosed as having 'neurogenic vomiting'. Having taken some western and Chinese drugs, the above symptoms were a little bit improved, but she would have nausea upon eating and with regurgitation. Because of the fear for vomiting, she did not dare to have food intake, with body weight reduction of 6 kilos in one month.     

Qiu Baoguo''''s Experience in Treating Consumptive Syndromes after Radio-Chemotherapies

Radiotherapy and chemotherapy are the important modern medical therapies for malignant tumors,yet they can also bring about serious local and systemic toxic side reactions so to decrease the patient;'s life quality,manifested by a series of consumptive symptoms.Having engaged in the combined work of Chinese and western medicine for nearly 50 years,the research fellow Qiu Baoguo in Henan Provincial Academy of TCM has developed his unique views on the TCM study of consumptive syndromes.The author of this essay had once the fortune tO follow Dr.Qiu in clinic,and specially would like to introduce in the following Dr.Qiu's experience in treating consumptive syndromes after radio-chemotherapies for patients with malignant tumor.     

Insomnia Due to Deficiency of Both the Heart and Spleen Treated by Acupuncture-Moxibustion and Chinese Tuina

OBJECTIVE: To observe therapeutic effects of the comprehensive therapy of acupuncture-moxibustion and Chinese Tuina for treatment of insomnia due to deficiency of both the heart and spleen. METHODS: 92 cases were divided randomly into the treatment group (treated by acupuncture-moxibustion and Chinese Tuina) and the control group (treated by acupuncture-moxibustion). RESULTS: The therapeutic effect of the treatment group was obviously superior to that of the control group (the CHI2 test showed P < 0.01). CONCLUSIONS: The comprehensive therapy of acupuncture-moxibustion and Chinese Tuina can give marked therapeutic effects for treatment of insomnia due to deficiency of both the heart and spleen.     

Analysis of CK-19,PDX-1,Nestin,Ngn3 in different donor islet purity in rats

Objective: To investigate if there are the CK-19, PDX-1, Nestin, Ngn3 positive cells in the donor islets of different purity in rats. Methods: Thirty male adult SD rats were randomly divided into 3 groups. Islets were isolated using digestion by ductal injection of collagenase. Group Ⅰ (n=10): Separating cell preparations were not purified, Group Ⅱ(n=10): Islet sediment was purified with 25% Ficoll400 ,Group Ⅲ (n=10): Islet sediment was purified with 25% and 11% Ficoll-400. The levels of protein of CK-19, PDX-1, Nestin and Ngn3 were detected by immunohistochemistry and the mRNA of CK-19, PDX-1, Nestin, Ngn3 was amplified by RT-PCR. Results: After two different purification methods applied, three islet preparations of different purities were obtained. The difference of islet purity was significant among various groups (P＜0.05). Compared with group Ⅱ and group Ⅲ,the protein and mRNA of CK-19, PDX-1, Nestin,Ngn3 were both higher in group Ⅰ; group Ⅲ was poorly expressed. Conclusions: The three different islet purity donor islet have different CK-19, PDX-1, Nestin, Ngn3 positive cells within them, indicating that there are some islet stem cells in the purified donor islet.     

A preliminary screening study on the associated proteins in human psoriasis vulgaris by serum proteomics technologies

Objective：To investigate the optimum screening conditions of associated proteins in human psoriasis vulgaris by serum proteomics technique, and to screen the different expression proteins related with psoriasis vulgaris. Methods：Serum samples of peripheral blood were collected from newly diagnosed psoriasis vulgaris patients in the clinic, and 20 matched healthy persons. Serum albumin IgG was removed by filtering with ProteoExtract Albumin/IgG. After comparative proteomics analysis the different protein spots were identified using 2-DE and MS. Results ：Electrophoresis figures with high resolution and reproducibility were obtained. Three different expression proteins were found only in the serum from psoriasis vulgaris patients, while nine other different proteins expressing from healthy volunteers. Conclusion：The protein expression was different in the serum between the psoriasis vulgaris patients and healthy volunteers. It was hoped that we could find the biomarkers related to psoriasis vulgaris by using proteomics.     

A CASE OF HUGE GASTRIC DISOPYROBEZOAR

PYTOBEZOARS are common bezoars in gastrontestinal tract, commonly seen in stomach and mall intestine,1but huge disopyrobezoars are     

Acupuncture Treatment of Chest Bi Syndrome

CASE HISTORY A male,a retired officer of 61 years old,paid his first visit on May 12,2006.The patient stated that 2 weeks before he suddenly got paroxysmal precordial choking pain,with irregular attacks.Each attack would last several seconds to several minutes.The attack was accompanied with heavy oppressed sensation in the precordial region,palpitation,lassitude,and in severe cases,sweating,which was once diagnosed by a hospital as'coronary heart disease'.Recalling the case history,the patient said that several days before he got angry with others because of some affairs in work,and he was not so used to the life pattern since he had just retired from his busy working post.At usual times,he was quick tempered and peevish.In recent days,he had poor appetite,and loose stool.     

Instructions to Contributors

The English edition of Chinese Journal of Integrative Medicine is edited and published by the Chinese Journal of Integrated Traditional and Western Medicine Press.Original scientific papers of advanced clinical and experimental medicine in the field of complementary/alternative medicine or integrative medicine are expected.     

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<正>As culture is the soul of a nation,the three books,"History of Chinese Philosophy,""History of Chinese Culture"and"History of Chinese Science and Technology"have demonstrated the superiority of our great Chinese culture,and its significant impact in the field of international culture.Both core value and practical value of the Chinese culture are very clear,reflected in the views of universe,nature,life,health,disease,and therapy and related considerations,concepts,and technical methods.