GasderminA3基因突变对小鼠毛囊β-catenin表达的影响

目的探讨GasderminA3(GSDMa3)基因突变前后β-catenin在毛囊中表达的变化。方法采用免疫组化S-P法、RT-PCR、Western blot等方法,检测出生后11d(毛囊生长期)、16d(毛囊退化早期)、18d(毛囊退化晚期)、24d(毛囊静止期)等各个时相点的GSDMa3基因突变鼠和C57BL/6(B6)鼠背皮毛囊β-catenin的表达表化。结果GSDMa3基因突变鼠和B6鼠β-catenin的表达均从11～24d逐渐减弱,但GSDMa3基因突变鼠β-catenin的表达在各个时期均强于B6鼠。在生长期,β-catenin表达于内外根鞘和毛母质细胞中,在GSDMa3基因突变鼠的表达强于B6鼠;在退化期,β-catenin主要表达于外根鞘和毛母质细胞中,在突变鼠的表达更强于B6鼠;在静止期,β-catenin主要表达在突变鼠的外根鞘细胞中,而在B6鼠的毛囊中表达很少。结论GSDMa3基因可能通过调节β-catenin的表达,影响毛囊的生长和发育。     

LSS基因敲除小鼠的饲养、繁殖及基因型鉴定

探讨羊毛固醇合成酶(LSS)基因敲除鼠的繁育和基因型分析.将引进杂合子小鼠进行饲养并繁育,提取其基因组DNA,利用PCR反应扩增目的 基因片段,鉴定出小鼠的基因型;Westem blot分析并比较LSS基因敲除杂合子小鼠和野生型小鼠的肝脏、皮肤组织中LSS蛋白的表达情况.成功的繁育出LSS基因敲除小鼠,使用PCR鉴定其子代小鼠包括野生型(LSS+/+,Wt)、杂合子(LSS+/-),未出现纯合敲除小鼠;Western blot结果表明:相较于野生型小鼠,LSS基因敲除杂合子小鼠在肝组织和皮肤组织中表达LSS蛋白量少;杂合子敲除鼠的生存能力比野生型小鼠弱.获得LSS基因敲除小鼠,PCR鉴定小鼠基因型具有简单、稳定的优点.     

毛囊周期性中肿瘤坏死因子受体1的表达

目的探讨肿瘤坏死因子受体1(TNFR-1)在毛囊周期中表达的规律及意义。方法采用石蜡切片免疫荧光、Western blot、RT-PCR检测小鼠毛囊生长期(出生后8、15 d,P8d、P15d)、退化期(出生后18 d,P18d)和静止期(出生后24 d,P24d)各时期TNFR-1的表达情况;免疫荧光检测毛囊细胞分化标记物AE13的周期性表达;TUNEL检测毛囊周期中的细胞凋亡情况。结果在4个时相点的皮肤中TNFR-1都存在不同强度的表达,RT-PCR与Western blot检测结果呈现出一致的规律,即从生长期中期(P8d)到生长期末期(P15d)呈现高表达趋势,退化期(P18d)和静止期(P24d)表达减弱并维持较低水平表达。免疫荧光的结果显示:TNFR-1表达在生长期主要表达于表皮层,毛囊的毛干和内根鞘以及毛母质,生长末期在外根鞘还出现明显表达;退化期在外根鞘、毛干及表皮层有表达,在次级毛芽出现很强的特异性表达;在静止期表皮层及杵状毛中呈现弱表达。AE13在生长期主要表达于表皮层以及毛干的皮质和毛小皮;退化期在毛干尤其是毛干基部端(即角质化区域和杵状毛形成区)强表达;在静止期弱表达于表皮层及杵状毛中。TUNEL检测凋亡结果显示:在生长期TUNEL阳性染色主要出现在表皮层,毛囊的毛干、内根鞘以及毛母质,生长末期还出现在外根鞘;在退化期出现在外根鞘、表皮层以及毛干基部端;在静止期P24d出现在表皮层、杵状毛、bulge区和皮脂腺。结论 TNFR-1表达于毛囊的各个时期,生长期中期到生长期末期呈现高表达趋势,退化期和静止期表达减弱并维持较低水平表达,其表达与毛囊细胞终末分化和凋亡有关。     

mTOR复合物组分Raptor和Rictor在小鼠毛囊周期中的表达与定位

目的：探讨哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)复合物组分Raptor和Rictor在小鼠毛囊周期中的表达与定位及其意义。方法：通过增殖标志物Ki-67的免疫荧光染色确定所用出生后不同时间的小鼠背部皮肤与所报道的毛囊周期阶段是否准确对应。采用real-time PCR技术检测Raptor和Rictor mRNA在出生后43 d (P43,静止期早期)、56 d(P56,静止期中期)、69 d(P69,静止期末期)、74 d(P74,生长期早期)中的表达情况;采用免疫荧光共染技术进一步检测Raptor和Rictor蛋白在上述各个时期中的表达与定位。结果：Ki-67免疫荧光结果表明所选取的小鼠皮肤与其所处毛囊周期阶段准确对应。Real-time PCR和免疫荧光检测结果表明：Raptor和Rictor mRNA在毛囊周期各时期的毛囊中表达差异无统计学意义(P>0.05);Raptor特异表达于毛囊隆突部的毛囊干细胞中,而Rictor主要表达在毛囊内根鞘中。结论：Raptor和Rictor的表达水平在毛囊周期各时期并未见明显差异,但Raptor会特异表达于毛囊干细胞中,而Rictor特异表达在内根鞘中,提示Raptor可能是毛囊干细胞的分子标志物之一,Rictor可能参与内根鞘的维持及毛干的形成。     

TNNT3(R69H)突变转基因小鼠模型的建立

目的 建立TNNT3(R69H)突变转基因小鼠模型.方法 构建pEGFP-TNNT3(R69H)转基因构件,通过原核显微注射方法将线性化、纯化后的外源质粒pEGFP-TNNT3(R69H)注射入BDF1小鼠受精卵中,胚胎移植至同期发情的假孕受体母鼠输卵管内,获得子代小鼠.用PCR和Southern blot方法检测子代鼠尾基因组DNA,通过RT-PCR及Western blot的方法检测TNNT3基因表达.结果 8只假孕小鼠共移植注射后的胚胎82枚,出生40只子代鼠,经PCR和Southern方法检测得到5只转基因阳性小鼠.对其子代小鼠进行RT-PCR、Western blot检测结果显示,TNNT3在转基因小鼠心脏和骨骼肌中表达量明显增多.结论 通过显微注射法使外源基因pEGFP-TNNT3(R69H)在小鼠基因组中整合,成功建立了TNNT3(R69H)突变转基因小鼠模型.     

MEK信号通路对H2O2诱导细胞角蛋白7在细胞中表达与定位的影响

目的细胞角蛋白7(Cytokeratin-7,Krt7)与肿瘤发展以及炎症反应密切相关,氧化应激在此过程中扮演着重要的角色,文中旨在通过构建小鼠Krt7的真核表达载体,并观察其在NIH 3T3细胞中的表达、定位以及在氧化应激反应下的变化情况。方法提取C57/BL6小鼠肺组织的总RNA,通过RT-PCR扩增得到Krt7编码序列,构建重组质粒pcDNA3-Krt7-HA。经脂质体转染NIH 3T3细胞,采用Western blot检测Krt7表达,免疫荧光检测其在细胞中的表达、定位以及在过氧化氢(H2O2)刺激下的变化情况。结果重组质粒构建成功。Western blot显示该质粒能够在NIH 3T3细胞中有效表达,免疫荧光检测表达产物主要分布在细胞质中,随着H2O2刺激时间的增加细胞内颗粒状蛋白聚集物的形成愈发明显且在60 min处发生入核现象。MEK通路抑制剂PD98059可明显抑制这一过程。结论成功构建了带HA标签的Krt7真核表达载体,该载体能在哺乳动物细胞中有效表达并正确定位,H2O2诱导Krt7在NIH 3T3细胞内的形态及定位变化依赖于MEK介导的信号通路。     

TPA促进小鼠毛囊中TCF3的表达

目的 检测12-O-十四烷酰佛波醋酸酯-13 (TPA)处理C57 BL/6( B6)小鼠背部皮肤后TCF3表达的变化情况.方法 采用免疫荧光染色、RT-PCR和Western blot等技术,检测TPA和丙酮分别处理7周大小的B6鼠背部皮肤1、2和4周后TCF3的表达情况.结果 与丙酮处理组相比,TPA处理组中B6鼠...     

Nestin转基因小鼠模型的建立及nestin基因在器官中的表达

目的构建人nestin基因表达载体,制备nestin过表达转基因小鼠。方法以人神经胶质瘤细胞株U251的cDNA为模板,分
3段扩增出nestin基因cDNA全序列,并将其克隆入含有强启动子ROSA26的pBROAD3载体,酶切鉴定、测序,除去原核序列,
纯化。显微原核注射建立转基因小鼠。PCR验证nestin基因整合,确定建立首建者。将阳性鼠传代。Western blot方法检测F3
代转基因小鼠与野生型小鼠主要器官（心、肺、脑、肾）的nestin 表达。结果测序结果证明成功构建了受ROSA26启动子调控的
nestin转基因载体。出生34只小鼠,PCR检测证实存在首建者2只。Western blot方法证实,与野生型小鼠相比,F3代转基因小
鼠脑、肺组织存在较高水平nestin 表达。结论首次成功建立nestin过表达转基因小鼠,外源基因可遗传到子代并在脑和肺组织
中稳定表达,为深入研究nestin在肿瘤转移、细胞“干性”的维持和细胞的分化等功能提供了良好的实验动物模型。
     

白血病细胞株KG-1中Mxi1基因突变的检测和表达载体的构建

目的 检测白血病细胞株KG-1中Mxil基因SID和bHLHzip区突变情况并构建Mxi1基因的野生型和突变型真核表达载体,观察其转染真核细胞后基因表达的情况。方法 应用RT-PCR和SSCP分析KG-1细胞株Mxil基因的突变,将Mxi1野生型/突变型基因cDNA插入到pDs-red2-N1质粒中,构建pDs-red2-N1/Mxi1(野生型/突变型)真核表达载体,采用脂质体方法将质粒转染COS-7细胞,流式细胞仪检测红色荧光蛋白表达并计算转染效率。RT－PCR检测转染后Mxi1基因的表达。结果 Mxi1基因突变位于Helix II区第82编码子的CAT/TAT(His/Tyr)。成功构建pDs-red2-N1/Mxi1(野生型/突变型)真核表达载体,经酶切、电泳可以看到在4700、1845bp存在两条电泳条带, Mxi1基因在COS 7细胞中得到表达,转染后COS-7细胞红色荧光蛋白表达的阳性细胞为18.07％,表达高峰出现在转染后第3天,并可持续表达6d左右。结论 Mxi1基因的高度保守区域bHLHzip存在基因突变,Mxi1突变基因的真核表达载体构建成功,并成功转染真核细胞COS-7     

丙型肝炎病毒F蛋白对p21基因的调节作用

目的研究丙型肝炎病毒(HCV)F蛋白对细胞p21基因转录、表达的影响。方法PCR扩增HCV 1b来源的F基因,克隆至pcDNA3.1真核表达载体。F基因质粒转染HepG2细胞,48h后抽提细胞总RNA和总蛋白,实时定量PCR及Western blot检测p21基因表达变化。结果HCV F蛋白在HepG2细胞内瞬时表达,相对于空载体对照(目标基因表达量为1),HCV F基因质粒转染细胞的p21转录水平为3.2,蛋白表达水平为1.4。结论HCVF蛋白增加p21转录和表达,其生物学效应值得进一步研究。     

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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