Increasing evidence has confirmed the existence of cancer stem cells (CSCs) in both hematological malignancies and solid tumors. However, the origin of CSCs is still uncertain, and few agents have been capable of eliminating CSCs till now. The aim of this study was to investigate whether bulk pancreatic cancer cells could convert into CSCs under certain conditions and explore whether metformin and curcumin can kill pancreatic CSCs. Aspc1, Bxpc3 and Panc1 pancreatic cancer cells were cultured in stem cell culture medium (serum-free Dulbecco's modified Eagle medium/Nutrient Mixture F-12 containing basic fibroblast growth factor, epidermal growth factor, B27 and insulin) for 5 days and it was found that all the pancreatic cancer cells aggregated into spheres and expressed pancreatic cancer stem cell surface markers. Then characteristics of Panc1 sphere cells were analyzed and cytotoxicity assays were performed. The results show that Panc1 sphere cells exhibited CSC characteristics and were more resistant to conventional chemotherapy and more sensitive to metformin and curcumin than their parent cells. These findings suggested that bulk pancreatic cancer cells could acquire CSC characteristics under certain conditions, which may support the "yin-yang" model of CSCs (interconversion between bulk cancer cells and CSCs). These results also showed that metformin and curcumin could be candidate drugs for targeting pancreatic CSCs.

Conditionally replicative adenoviruses (CRAds) are widely used for cancer biotherapy and show a significant growth-suppressing effect on many types of cancer. However, it was reported that breast cancer was highly resistant to the infection of traditionally used adenovirus of serotype 5 (Ad5)-based CRAds. Although partial substitution of the fiber protein of replication-deficient Ad5 with that of adenovirus of serotype 35 (Ad35) facilitated infection of breast cancer cells by adenoviral vectors, it is still unknown whether this modification can improve CRAds in their tumor-eliminating capacity. We generated a 5/35 fiber-modified CRAd with a p53 cDNA construct and investigated whether this alteration in fiber region can make CRAds suppress the growth of breast cancer more effectively. Our data reinforced the proposal that 5/35-modified fiber conferred higher adenovirus infectivity for breast cancer cells than natural Ad5 fiber. Interestingly, 5/35 fiber-modified CRAd replicated more efficiently in breast cancer cells than Ad5-based CRAd. We also found 5/35 fiber-modified CRAd mediated higher expression of p53 in breast cancer cells. In vitro, 5/35 fiber-modified CRAd eliminated breast cancer cells more efficiently. Growth of xenograft tumors in nude mice was also significantly retarded by 5/35 fiber-modified CRAd. The 5/35 fiber-modified CRAd suppressed the growth of breast cancer cells more effectively than Ad5-based CRAd, both in vitro and in vivo. Thus CRAd with 5/35 hybrid fiber may be a promising vector for breast cancer treatment.

Objective To express the recombinant human canstatin protein, and to examine its biological activity. Methods Canstatin cDNA was cut off from the plasmid pUCm-T/canstatin with restriction enzymes BamHⅠ and Hind Ⅲ. The cDNA fragment was then ligated into the correspondence sites of plasmid pET-22b(+) by T4 DNA ligation enzyme and transformed into E.coli BL21 which was induced to express proteins with isopropyl-1-thio-b-dgalactopyranoside (IPTG). The expressed proteins were analyzed by SDS-PAGE and purified through Ni-NTA column affinity chromatography. Chick chorioallantoic membrane (CAM) assay was performed to determine the activity of the recombinant protein. Results Canstatin cDNA from pUCm-T showed one clear objective DNA band with electrophoresis. Seven of positive colonies were selected and identified by restriction enzyme analysis with BamH Ⅰ and Hind Ⅲ. Electrophoresis revealed that all selected colonies had two specific bands,one near the location of primary plamid,the other near that of objective gene fragment. After IPTG induction, there was a new protein band about 24 000 on SDS-PAGE.The induced product over total bacterial proteins in 1,2, 3. and 4 hours after induction was 18. 2%, 18. 8%, 23.0% and 23.4%, respectively, by densitometry examination. CAM assay demonstrated that the recombinant canstatin protein significantly inhibited the embryonic neovascularization in a dose-dependent manner. Conclusion The prokaryotic expression vector of human canstatin gene has been successfully constructed, laying the foundation for further clinical study.

AIM:To examine the effect of canstatin, a newly discovered endogenous inhibitor of angiogenesis, in the treatment of pancreatic cancer in vivo.METHODS:The canstatin cDNA fragment was synthesized and amplified from the total RNA extracted from human placenta tissues by RT-PCR. The resulting product was firstly cloned into pUCm-T vector, then into plasmid pET-22b (+) and transformed into E. coli BL21. Isopropyl-1-thio-b-Dgalactopyran-oside (IPTG) was used to induce the expression of canstatin protein and affinity chromatography was used to purify the protein. To determine the activity of purified recombinant human canstatin (rhCanstatin), orthotopic xenograft human pancreatic cancer models were established. Human pancreatic cancer cells (SW1990) were injected into the pancreas of BALB/c nude mice. Twenty-four nude mice with orthotopic xenograft tumor were randomly divided into 3 groups 10 d after the inoculation, and were treated with PBS 0.3 mL, or canstatin 5 mg/kg, or 10 mg/kg per day for 3 wk intraperitoneally. When the experiment was over, all tumors were resected and the effects of rhCanstatin on tumor growth, microvessel density (MVD) were analyzed.RESULTS:After IPTG induction, SDS-PAGE showed a new monomeric 24 kDa protein band. This protein was purified through affinity chromatography and refolded through dialysis with a final concentration of 60 mg/L. In orthotopic pancreatic cancer models, the final tumor volume in groups treated with PBS, canstatin 5 mg/kg, 10 mg/kg were 355.21+/-39.54 mm3, 112.73+/-10.47 mm3, and 61.75+/-6.99 mm3 respectively. The immunohistochemical examination showed that the MVD in tumors treated with canstatin was significantly less than that in other group.CONCLUSION:These findings demonstrate that the rhCanstatin effectively retards the growth of pancreatic cancer in a dose-dependent manner through inhibiting angiogenesis and may be a promising therapeutic agent for pancreatic cancer treatment in the clinic.

Objective: To construct prokaryotic expression vector of human canstatin gene,express and purify its recombinant protein. Methods: The total RNA was extracted from human placenta tissues.The canstatin gene fragment was amplified from the total RNA by RT-PCR.The resulting product was cloned into pUCm-T vector and was sequenced.Then the confirmed canstatin cDNA was cloned into plasmid pET-22b(+) and transformed into E.coli BL21 where it was induced to express proteins by isopropyl-1-thio-b-Dgalactopyranoside(IPTG).The expression of protein was analyzed through SDS-PAGE.Cells induced 3 hours by IPTG were harvested,sonicated briefly and the proteins were purified through affinity chromatography. Results: The target sequences were specifically amplified through RT-PCR,cloned into pUCm-T vector,and then transformed into E.coli DH5α.Six white colonies were selected and cut by BamHⅠ and Hind Ⅲ separately.The plasmids of one white colony showed positive results and was sequenced,demonstrating to be the same as that of canstatin gene in GenBank.Then canstatin cDNA was cut down from pUCm-T,ligated into the vector pET-22b(+) and then transformed into(E.coli) BL21.After an overnight incubation,seven white colonies were picked and showed positive results after digested by both BamHⅠ and Hind Ⅲ.After IPTG induction of one positive colony,a new protein band about Mr 24 000 showed on SDS-PAGE.The percent expressed product over total bacterial proteins after 1,2,3,and 4 hours of induction was 18.2%,18.8%,23.0% and 23.4% respectively estimated by densitometry.After affinity chromatography,SDS-PAGE showed only one clear band existed in 125 mmol/L or 250 mmol/L imidizone elution. Conclusion:The prokaryotic expression vector of human canstatin gene has been successfully constructed.Further more,purified recombinant proteins are obtained through affinity chromatography,laying foundation for further study of its clinical use.

OBJECTIVE:To clone human canstatin gene and express its recombinant protein.METHODS:The total RNA was extracted from human placenta. The canstatin gene fragment was synthesized and amplified from the total RNA by RT-PCR. The resulting product was cloned into pUCm-T vector and transformed into E.coli DH5alpha through electroporation. The gene was sequenced by the Sanger Dideoxy-mediated chain-termination method, and then the canstatin cDNA was cloned into the BamHI and HindIII sites of plasmid pET-22b (+) and transformed into E.coli BL21 where it was induced to express proteins by isopropyl-1-thio-b-Dgalactopyranoside (IPTG).RESULTS:The extracted total RNA was separated into three clear bands indicating 28S, 18S, and 5S after electrophoresis. The canstatin gene fragment was synthesized and amplified from the total RNA by RT-PCR. The resulting products were cloned into pUCm-T vectors, and then were transformed into E.coli DHSa. After an over night culture, both blue and white colonies were found on the agar plate. Six white colonies were selected and cut by BamHI and HindIII. The plasmids DNA in one white colony showed one band near the location of primary plasmid after digested by BamHI and two bands near the locations of primary plasmid and objective gene fragment after digested by HindIII. The cloned gene in this white colony was sequenced and demonstrated to have the same sequence as that of canstatin gene in GenBank. Then canstatin cDNA was cut down from pUCm-T with BamHI and HindIII and ligated into the vector pET-22b (+). The resultant plasmid pET-22b (+)/canstatin was then transformed into E.coli BL21. White colonies were found on LB agar plate. Seven of them were selected and their plasmids were digested with both BamHI and HindIII. After electrophoresis, all selected colonies showed two specific bands, one was found near the location of primary plasmids, and the other near that of objective gene fragment. After IPTG induction, there was a new protein band about Mr 24 000 on SDS-PAGE. As estimated by densitometry, the percentage of the expressed product over total bacterial proteins was 18.2%, 18.8%, 23.0% and 23.4%, respectively, 1, 2, 3, and 4 hours after induction.CONCLUSION:Human canstatin gene was successfully cloned and its recombinant proteins were expressed in this study.

胰腺癌恶性程度高,预后差,严重威胁着人类健康.近几年,随着分子生物学的迅猛发展,胰腺癌研究在多方面取得重要进展,此文就其流行病学、病因、发病机制、诊断和治疗等各方面的研究热点与难点作一综述.

目的:克隆血管能抑素canstatin基因,测定并分析其基因序列.方法:从人胎盘组织中提取总RNA,经逆转录-聚合酶链式反应（RT-PCR）扩增出canstatin基因,克隆进载体pUCm-T获得重组质粒pUCm-T/canstatin,转化E.coliDH5α,挑选出阳性克隆,测定基因序列.结果:从人胎盘组织中提取出RNA,10g/L琼脂糖凝胶电泳显示3条清晰条带,分别对应28S,18S,5S.分光光度计测定总RNA的A260=0.879,A280=0.410,A260:A280=2.095,总RNA浓度为1.8g/L.RT-PCR扩增产物与预期的目的基因canstatin长度一致.RT-PCR产物与载体pUCm-T连接过夜后,转化E.coliDH5α,在LB平板上生长出蓝色和白色菌落.挑选6个白色菌落做酶切鉴定,其中一个菌落经BamHI酶切后,只出现1条特异性条带,位置与酶切前质粒相近,而经HindⅢ酶切后,出现2条特异性条带,其中1条与酶切前质粒位置相近,另1条与目的基因位置基本一致,证实为阳性克隆.测定该阳性克隆基因序列,结果显示其与Genbank中公布的canstatin基因序列完全一致.结论:成功克隆了canstatin基因,为进一步研究其抗肿瘤作用打下基础.

胰腺癌是消化系统常见恶性肿瘤,恶性程度高,侵袭性强,预后差,死亡率接近于发病率.尽管近50 a,各种传统的治疗手段如手术,放疗,化疗或这些方法的联合应用取得较大进展,但并未明显改善胰腺癌患者的预后.近30 a研究证实血管生成是许多肿瘤包括胰腺癌生长转移过程中的关键步骤,抗血管生成治疗,尤其是抗血管生成基因治疗作为胰腺癌治疗新视野,为改善胰腺癌预后带来了新的希望,成为研究的热点.本文就该方面的研究进展做一综述.

编者按本文是消化内镜在常见临床情况下应用的系列讨论之一,由美国消化内镜学会提供.在撰写这一指南的过程中,除MEDLINE检索到的文章外,还参考一些专家推荐的文章.内镜的合理应用指南是基于目前的一些重要的综述和专家共识.还需要大量的临床对照研究加以确定和必要的修订.临床上遇到情况和指南有所差异时应适当调整.