Expression, purification and characterization of anti-α2δ1/NanoLuc fusion protein / 生物工程学报

The existence of cancer stem cells is regarded as the major cause for therapeutic resistance and relapse of a variety of cancer types including hepatocellular carcinoma (HCC). However, the tracing of such a subpopulation in vivo has been challenging. We have previously demonstrated that the isoform 5 of the voltage-gated calcium channel α2δ1 subunit, which can be recognized specifically by a monoclonal antibody 1B50-1, is a bona fide surface marker for HCC stem cells. Here we developed a strategy for optical imaging of α2δ1-positive cells by using a fusion protein containing the single chain variable fragment (scFv) of Mab1B50-1 and the luciferase NanoLuc which was tagged with Flag in the C-terminal. The scFv of Mab1B50-1 was fused to the N-terminal of NanoLucFlag using overlap PCR, and the recombinant fragment, which was named as 1B50-1scFv-NanoLucFlag, was subsequently cloned into a eukaryotic expression vector. The resulting construct was transfected into FreeStyle 293F cells in suspension using PEI reagent. The expression of the fusion protein was identified as a protein with molecular weight about 50 kDa by Western blotting. After purification by ANTI-FLAG® M2 affinity chromatography, 1B50-1scFv-NanoLucFlag was demonstrated to bind to α2δ1 positive cells specifically with a Kd value of (18.62±1.84) nmol/L. Furthermore, a strong luciferase activity of 1B50-1scFv-NanoLucFlag was detected in α2δ1 positive cells following incubation with the fusion protein, indicating that the presence of α2δ1 could be quantified using this fusion protein. Hence, 1B50-1scFv-NanoLucFlag provides a potential tool for optical imaging of α2δ1 positive cancer stem cells both in vitro and in vivo.

A Pharmacodynamical Study on the Improvement of Senile Drosophilas with Fragmented Sleep with the Administration of Shuang Xia Decoction / 世界科学技术-中医药现代化

This study aimed at unfolding the therapeutic effects of freeze-dried powder separated from Shuang Xia decoction (SXD) on female senile drosophila melanogaster with fragmented sleep.Taking drosophilas as the model organisms,the locomotor activity was monitored using the autonomic monitoring software to explore the regulation of fragmented sleep in senile drosophilas by the treatment of SXD.As a result,it was found that the optimum concentration of SXD was 2.50％,while the desirable therapeutic duration was 4 days.In comparison with the control group,35-day-old virgin drosophilas'sleep was prolonged in the SXD group and the positive control group.The positive drug mainly affected their sleep in the daytime,while SXD impacted their sleep at night featuring the prolonged fragmented sleep.In conclusion,it was demonstrated that the freeze-dried powder of SXD effectively alleviated intermittent insomnia in the female senile drosophilas.Compared with positive drug,SXD also mitigated intermittent insomnia at night without significant changes in the sleep of the drosophilas in the daytime.Above all,SXD was beneficial in the regulation of sleep-wake rhythm in the drosophilas.

Research on Promotion of Wound Healing by Amputated lumbricus Extract / 世界科学技术-中医药现代化

On the basis of pre-experiment research and the hypothesis of“amputated lumbricus”, this research was aimed to explore mechanism of active components of the amputated lumbricus to promote wound healing. Skin excision was used to establish the mice model. The amputated lumbricus extract was prepared. HE staining and immunohistochemistry techniques were used in the determination of the wound healing rate and changes of VEGF, bFGF, TGF-β1 expression during wound healing period. The results showed that compared with the blank control group, the healing rate of the amputated lumbricus extract group was better. And the HE staining showed better improvement of traumatic tissues. There was no statistic differences on the expression of VEGF and TGF-β1 between the amputated lumbricus extract group and the normal saline group (P> 0.05). The expression of bFGF in amputated lumbricus extract group reached peak earlier than the control group and also lasted a longer time. The amputated lumbricus extract group reached peak on the first day, which had a significant difference (P < 0.05) compared with the control group at the same timepoint. It was concluded that the external application of amputated lumbricus extract had wound healing effect on traumatic skin of mice. Its mechanism may be irrelevant to the expression of VEGF and TGF-β1. However, it may be related to the increasing of bFGF expression in the injured regions during the inflammation stage and proliferation stage.

Construction of a lentivirus vector for RNA interference of myosin Va and its effect on motility and migration of PG cells / 解剖学报

Objective To construct a lentivirus vector for RNA interference targeting myosin Va gene and to observe its effect on motility and migration of human pulmonary giant cell carcinoma PG cells. Methods Based on the efficient target sequence for myosin Va RNAi, two pairs of oligo DNA containing myosin Va RNAi target sequence or scramble sequence were synthesized and inserted into pSuper vector, followed by sequence analysis. The expressing cassette H1 promoter-shM5A/shCON from the recombinant pSuper plasmid was then transferred to the lentivirus vector plenti4, and the recombinant lentivirus was packaged. PG cells were transduced with the packaged lentivirus and the positive cells were screened by zeocin selection. RT-PCR was performed to determine the myosin Va RNAi efficiency in zeocin-resistant PG cells, and wounding assay and Boyden chamber assay were utilized to examine the capabilities of motility and migration in myosin Va RNAi PG cells. Results Restriction enzyme digestion and sequencing confirmed the successful construction of the lentivirus vector containing myosin Va RNAi target or scramble sequence. RT-PCR result showed that myosin Va mRNA levels were remarkably reduced in lentivirus-based myosin Va RNAi PG cells. The abilities of motility and migration were also significantly inhibited in lentivirus-based myosin Va RNAi PG cells, as demonstrated in wounding assay and Boyden chamber assay.Conclusion Myosin Va RNAi lentivirus vector was successfully constructed and efficiently repressed myosin Va expression in PG cells. Repression of myosin Va by RNAi led to the inhibition of PG cells motility and migration, indicating that there might exist correlation between the expression of myosin Va and cancer progression.

Development and Application of a Biofeedback Knee Joint Rehabilitative Device with Software Games for Anterior Cruciate Ligament Reconstruction / 中国康复理论与实践

@#: Objective To develop a rehabilitation device and software game, and provide the system for evaluation and training of anterior cruciate ligament (ACL) reconstruction.MethodsThe development and clinical application of a biofeedback knee joint movement rehabilitation system for ACL reconstruction were very important.ResultsBIOKS readings reproduce Zebris 3D motion analysis system measurements reliably between 0° and 150° ( P>0.05). Significant knee joint movement training effect ( P<0.05 ) in JPS assessment. However, there were no differences ( P≥0.05) between SST and KAS assessments.ConclusionThe improvement of joint position sense and body centroid sway under single leg support conditions.

Downregulation of Raf kinase inhibitory protein in human lung cancer cell lines / 基础医学与临床

Objective To detect the expression level of RKIP in human lung cancer cell lines and to investigate the relationships between RKIP and cell metastasis. Methods The expression level of RKIP in human lung cancer cell lines was analyzed by RT-PCR. To construct and express human RKIP fusion protein and to immunize New Zealand rabbit with purified fusion protein to prepare polyclonal antiserum.The antiserum was titered by ELISA. The specifcity of antiserum and expression level of RKIP in human lung carcinoma cell lines was analyzed by western blotting. Results The fusion protein was successfully expressed. The prepared polyclonal antibody reacted specifically with RKIP in human cells. Downregulation of RKIP was detected in three lung cancer cell lines with metastasis, and it is more significant in A549 with higher metastasis.Conclusion The RKIP antiserum with high titer and good specificity could meet the requirement for studying on RKIP. Downregulation of RKIP has correlation with human lung carcinoma cell metastasis.

Selective heterologous communication among human lung epithelial cells, fibroblasts, and lung cancer PG cells / 北京大学学报(医学版)

Objective: To further understand the roles of gap junctional intercellular communication in carcinogenesis and progression of human lung cancer. Methods: The heterologous communication was characterized among human lung epithelial cells HLEC, human lung fibroblasts HLF, human lung giant carcinoma PG cells and its Cx43 transfectants PG/C4 by using a preloading assay. Cx43 immunofluorescent staining and Northern blot were performed to examine Cx43 gene expression. Results: Although both human lung firoblasts HLF and epithelial cells HLEC expressed Cx43 and had very strong homologous communication, HLEC cells were unable to communicate with HLF cells. The human lung carcinoma cell line PG was defective of both homologous communication, and heterologous communication with its epithelial origin HLEC. Transfection of Cx43 into PG cells, which rescued PG cells' homologous communication and enhanced heterologous communication with HLF cells, could restore heterologous coupling with HLEC cells. Conclusion: Selective heterologous communication exists among different kinds of human lung cells. when human lung carcinoma cells lost coupling with its epithelial origin, Cx43 gene expression is not enough to establish heterologous communication between them.

Construction of a recombinant adenovirus containing MMP-9 signal peptide and PEX domain of human MMP-2 and study of its activity / 中国肿瘤生物治疗杂志

Objective: To construct a recombinant adenovirus (abbreviated as ET-M9-PEX) containing MMP-9 signal peptide and noncatalytic carboxyl-terminal hemopexin domain of human MMP-2, and to use the constructed adenovirus as a drug bioreactor in vivo to enhance the expression of an anti-angiogenesis factor for treatment of tumor by a gene therapy strategy. Methods: Adenovirus vector containing M9-PEX gene was constructed by PCR and gene recombination, and was packaged and amplified in L293 cells to obtain ET-M9-PEX recombinant adenovirus with infective ability. The expression and secretion of PEX in ET-M9-PEX-infected cells were detected by Western-blotting and immunofluorescent staining. The inhibitory effect of ET-M9-PEX-conditioned medium on EC cells proliferation was detected by growth curve and its inhibitory effects on angiogenesis and tumor growth were determined by chicken chorioallantoic membrane (CAM) assay in vivo. Results:ET-M9-PEX was successfully constructed and the expression and secretion of PEX in ET-M9-PEX-infected cells were verified. The ET-M9-PEX conditioned medium significantly inhibited the proliferating rate of EC cells. The tumor weights from ET-M9-PEX-infected PG cells in CAM and gradeⅠvessel number were reduced by 57.57%(P

EXPRESSION OF GST-SNAIL FUSION PROTEIN IN PROKARYOTIC CELLS AND PREPARATION OF POLYCLONAL ANTIBODY AGAINST SNAIL / 解剖学报

Objective The aim of this study was to prepare anti-snail polyclonal antibody and make it widely useful in snail detection. Methods The DNA fragment encoding human full length 264 amino acid of snail was obtained by PCR from cDNA library of human umbilical vein epithelial cells and was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST).The GST-snail fusion protein was expressed by E.coli BL21 after IPTG induction and purified from total proteins of BL21 transformed by the recombinant plasmid pGEX-4T-1/snail.The New Zealand rabbit was immunized with the purified fusion protein to prepare polyclonal antiserum.The antiserum was identified by western blotting and immunofluorescent staining. Results The prokaryotic expression plasmid pGEX-4T-1/ snail was successfully constructed,and the fusion protein GST-snail was expressed efficiently.The polyclonal antibody raised in the rabbit could react specifically with snail in human cells.Conclusion The snail antiserum was of good purity with high titer and specificity which could satisfy the requirement for studying immuno-analysis on snail protein.