人CD44基因真核表达载体构建及在乳腺癌细胞中的表达

目的:从人类乳腺癌耐多柔比星细胞株细胞(MCF-7/Adr)中克隆CD44基因并构建CD44基因真核表达载体pcDNA3.1-CD44;将pcNDA3.1-CD44稳定转染入人乳腺癌细胞株(MCF-7)细胞中.方法:从MCF-7/Adr细胞中提取总RNA进行RT-PCR,获得全长的cDNA模板;将含有CD44基因的cDNA模板使用限制性内切酶进行双酶切获得带有酶切位点的CD44基因,再将带有酶切位点的CD44基因经TA克隆后测序验证,然后亚克隆入真核表达载体pcDNA3.1中,双酶切鉴定后再次测序验证.脂质体法将pcDNA3.1-CD44质粒转染到MCF-7细胞中,48小时后RT-PCR和流式细胞术检测转染前后CD44基因、蛋白在MCF-7细胞的表达变化.结果:成功从人MCF-7/Adr细胞中克隆获得CD44基因并构建真核表达载体,转染后在MCF-7中检测到CD44基因mRNA和蛋白水平表达上调;将上述表达CD44基因及蛋白的瞬时转染MCF-7细胞经G418筛选两周后获得阳性克隆.结论:成功克隆和建立人CD44基因真核表达质粒;pcDNA3.1-CD44能在MCF-7细胞中表达;在MCF-7/Adr中新发现一种CD44基因的变异体(基因库号FJ216964);这为进一步研究其基因功能打下了基础.     

重组肝细胞生长因子质粒的构建及其在成骨细胞中的表达

目的 构建人肝细胞生长因子(hHGF)真核表达质粒,探讨重组质粒对体外培养胎兔成骨细胞增殖分化的影响,为转基因治疗骨科疾病提供实验基础.方法 RT-PCR扩增人肝脏中hHGF全长cDNA片段,克隆入pcDNA3.1( )真核表达载体中,用脂质体法将pcDNA3.1( )-hHGF质粒转染成骨细胞,通过G418筛选获得阳性克隆,用免疫荧光染色检测hHGF基因在成骨细胞内的表达;MTT法和FCM检测pcDNA3.1( )-hHGF转染后对细胞增殖和细胞周期的影响;采用RT-PCR和ELISA方法检测hHGF在成骨细胞中的表达.NPP法检测碱性磷酸酶合成情况.结果 成功构建hHGF真核表达质粒,免疫组化和RT-PCR检测显示转染成骨细胞后细胞内hHGF mRNA呈现高水平表达;MTT法和FCM检测显示重组质粒转染后能促进成骨细胞的增殖,S期细胞比例增多;ELISA法检测到hHGF在细胞中有分泌性表达.转染重组质粒的成骨细胞合成碱性磷酸酶能力显著提高.结论 成骨细胞经基因转染后可以表达hHGF,外源性hHGF基因能够刺激成骨细胞增殖、分化及成骨活性.     

pcDNA3.1(+)-CYP19-GFP真核表达质粒构建

目的:构建pcDNA3.1(+)-CYP19-GFP、pcDNA3.1(+)-CYP19W39R-GFP、pcDNA3.1(+)-CYP19R264C-GFP与pcDNA3.1(+)-CYP19W39R-R264C-GFP真核表达质粒,并观察pcDNA3.1(+)-CYP19-GFP质粒在MCF-7和Bcap-37细胞中的表达。方法:①采用RT-PCR技术扩增CYP19 cDNA,插入pcDNA3.1(+)载体中,构建pcDNA3.1(+)-CYP19表达质粒;酶切pcDNA3.1(+)-CYP19(304bp BamHⅠ)-GFP和pcDNA3.1(+)-CYP19质粒,构建pcDNA3.1(+)-CYP19-GFP表达质粒;②以pcDNA3.1(+)-CYP19-GFP质粒为模板,采用定点突变技术,构建pcDNA3.1(+)-CYP19W39R-GFP和pcDNA3.1(+)-CYP19R264C-GFP及pcDNA3.1(+)-CYP19W39R-R264C-GFP表达质粒;③pcDNA3.1(+)-CYP19-GFP质粒通过脂质体介导转染MCF-7和Bcap-37细胞,荧光显微镜观察其在细胞中的表达。...     

人纤溶酶原K5区基因真核表达载体的构建和活性鉴定

目的 构建含人纤溶酶原K5区基因的真核表达载体，转染人乳腺癌细胞株MDA—MB—231，观察阳性克隆表达的K5蛋白对人脐静脉内皮细胞株ECV304和MDA—MB—231细胞增殖的影响。方法 应用PCR将人纤溶酶原信号肽序列引入K5cDNA，所得的目的片段与真核表达载体pcDNA3重组，构建重组质粒poDNA3K5，脂质体法将其转染MDA—MB—231，G418筛选阳性克隆，PCR鉴定，RT-PCR和Western blot检测K5的表达。将鉴定正确的阳性克隆的培养上清作用于ECV304细胞，MTT、法检测其增殖情况，并用MTT法检测转染pcDNA3K5对MDA—MB—231细胞增殖的影响。结果 构建的重组质粒pcDNA3K5经酶切鉴定、测序正确，将其转染MDA-MB-231后挑取的阳性克隆有3个经PCR鉴定正确，并经RT-PCR和Western blot检测证实K5的表达。阳性克隆的培养上清作用于ECV304细胞后，其存活率降低；转染pcDNA3K5对MDA—MB—231细胞增殖无明显影响。结论 应用脂质体法将带有人纤溶酶原信号肽序列的K5cDNA转染MDA-MB-231细胞后，其分泌产生的有生物学活性的K5，呈现对ECV304细胞增殖的抑制作用，而对MDA-MB-231细胞的生长则无影响。     

人CIDE3基因克隆和真核表达载体构建及促凋亡作用的研究

目的：克隆人诱导细胞死亡的DFF45样效应因子3（CIDE3）全长基因,构建真核表达质粒pcDNA3.1（＋）-CIDE3,并检测其促凋亡作用.方法：①提取人腹部皮下白色脂肪组织的总RNA,经RT-PCR获得CIDE3全长基因,克隆入真核表达载体pcDNA3.1（＋）,构建重组质粒pcDNA3.1（＋）-CIDE3,并进行序列测定.②用脂质体法将pcDNA3.1（＋）-CIDE3转染人宫颈癌细胞系HeLa细胞,通过TUNEL法检测细胞凋亡情况.结果：①经RT-PCR扩增得到特异性717 bp的目的片段,构建的重组质粒pcDNA3.1（＋）-CIDE3经序列测定,结果与Genbank发表的序列完全一致.②转染Hela细胞后,经Tunel染色法检测发现,转染pcDNA3.1（＋）-CIDE3后有大约25%的细胞发生了凋亡,与转染空质粒pcDNA3.1（＋）（＜5%）及未转染质粒的细胞相比,其凋亡细胞明显增多.结论：成功克隆了人CIDE3全长基因,构建了真核表达质粒pcDNA3.1（＋）-CIDE3,并验证了CIDE3基因可诱导细胞发生凋亡的生物学作用,为进一步研究CIDE3的生物学功能奠定了坚实基础.     

人HNF1α真核表达载体的构建及表达

目的 构建人肝细胞核因子-1α(hepatocyte nuclear factor 1α,HNF1α)真核表达载体,在体外表达并检测.方法 提取HepG2总RNA,用RT-PCR方法制备HNF1α cDNA,进行T-A 克隆.Hind Ⅲ/EcoR Ⅰ双酶切测序正确的重组质粒,回收HNF1α cDNA片段,将其亚克隆于pcDNA3.1( )载体的多克隆位点内.将构建好的真核表达质粒用脂质体法转染HepG2细胞,RT-PCR和免疫印迹法检测HNF1α的表达. 结果正确构建了pcDNA3.1( )-HNF1α重组载体,并且在转染该质粒的HepG2细胞中检测出了HNF1α的高表达. 结论成功地构建了人HNF1α真核表达载体,其可以在体外表达.     

pcDNA3.1- Bcl-2真核表达载体构建

目的 克隆大鼠Bcl-2基因（B cell lymphoma）全长cDNA,构建含大鼠Bcl-2基因的重组真核表达质粒。方法 采用RT-PCR的方法从大鼠脾脏组织中扩增全长Bcl-2cDNA,将扩增产物克隆至pMD18-T载体, 测序证实后,再克隆至真核表达载体pcDNA3.1(+),构建了pcDNA3.1- Bcl-2重组质粒。结果: RT-PCR获得长度为720 bp的阳性产物,经T载体和pcDNA3. 1(+)真核表达载体克隆、酶切鉴定及序列分析后,证实真核表达质粒pcDNA3.1- Bcl-2构建成功。结论 成功克隆了Bcl-2基因,并构建了pcDNA3.1- Bcl-2重组质粒,为进一步研究Bcl-2基因的功能及应用Bcl-2进行基因治疗奠定基础。     

嗜肺军团菌PAL抗原基因的克隆及真核表达

目的 构建真核表达重组质粒pcDNA3.1-PAL,并观察其在NIH3T3细胞中的表达.方法.结论PCR扩增嗜肺军团菌PAL基因,定向克隆入真核表达载体pcDNA3.1( )中,将经酶切、PCR扩增及序列测定正确的重组质粒命名为pcDNA3.1-PAL.脂质体法将重组质粒pcDNA3.1-PAL转染NIH3T3细胞,经免疫荧光和RT-PCR检测其瞬时表达和稳定表达产物.结果 重组质粒pcDNA3.1-PAL转染NIH3T3细胞后获得了有效表达.结论 成功构建了真核表达重组质粒pcDNA3.1-PAL为嗜肺军团菌核酸疫苗的进一步研究奠定了基础.     

人类TSLC1基因克隆及真核表达载体的构建

目的:克隆人类TSLC1基因并构建其真核表达载体pcDNA3.1( )/TSLC1.方法:从正常皮肤组织中提取总RNA,采用RT-PCR方法扩增TSLC1基因全长cDNA,克隆入pMD18-T载体并转化大肠杆菌JM109,经PCR、酶切鉴定均为阳性的克隆,进行核苷酸测序分析,再将TSLC1基因定向克隆入pcDNA3.1( )载体中构建表达载体.结果:RT-PCR扩增后的产物在约1413 bp处出现明显的特异性条带,TSLC1基因的cDNA片段被成功插入真核表达载体pcDNA3.1( )质粒的多克隆位点,经鉴定与GenBank收录的TSLC1 cDNA 序列一致.结论:TSLC1基因的cDNA片段被成功克隆,为以后的研究奠定了基础.     

人Endostatin cDNA的克隆及其在HT1080细胞中的表达

目的：构建能表达人Endostatin的真核表达质粒。方法：设计引物从成人肝cDNA文库中通过PCR扩增到Endostatin cDNA，并在5′端融合编码人胰岛素领带肽序列，经测序证实后连接到真核表达载体pcDNA3.1(-)中，将携带基因的真核表达载体质粒pcDNA3.1(-)/SEND转染HT1080细胞后用G418筛选获得克隆，采用RT-PCR和West-em-blot检测Endostatin的表达。结果：测序结果表明克隆的人EndostatincDNA完全正确，并且在其5′端融合编码人胰岛素信号肽序列；RT-PCR显示转染后的HT1080细胞可以有效地转录EndostatinmRNA；Westem-blot检测到转染后的HT1080细胞上清有相应的蛋白质，大小为20kD左右。结论：含有人胰岛素信号肽序列的人EndostatincDNA重组质粒pcDNA3.1(-)/SEND转染HT1080细胞后可以有效的表达目的蛋白并能分泌到细胞外，本研究为Endostatin的功能研究及应用于基因治疗提供基础。     

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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