ObjectiveTo obtain recombinant H.pylori adhesin A(rHpaA)by molecular cloning,protein expression and purification,immunize BALB/c mice to prepare anti-HpaA polyclonal antibody,and analyze its antibody specificity.MethodsThe three-dimensional structure and antigenic properties of rHpaA were analyzed by bioinformatics softwares such as Phyre2 and DNAstar;Adhesin HpaA gene was obtained by PAS(PCR-based accurate synthesis)and inserted into plasmid pCzn1.The prepared recombinant plasmid pCzn1-rHpaA was transformed to E.coli Artic Express(DE3),induced by IPTG and purified by Ni-IDA affinity chromatography to obtain rHpaA protein,which was identified for reactivity by Western blot.Six male BALB/c mice were immunized with rHpaA plus Freund's adjuvant to prepare anti-HpaA polyclonal anti-body,and the antibody specificity was identified by ELISA.ResultsrHpaA showed good three-dimensional structure and antigenic properties.Restriction analysis and gene sequencing showed that the recombinant plasmid pCzn1-rHpaA contained completely correct HpaA gene sequence.The recombinant strain pCzn1-rHpaA/Arctic Express expressed the soluble target protein rHpaA,which accounted for about 68.3% of total protein in the supernatant,with a purity of 98.1%.rHpaA bound to anti-His antibodies and anti-H.pylori antibodies;The anti-HpaA polyclonal antibody specifically recognized rHpaA and H.pylori lysates.ConclusionrHpaA protein with high purity can be obtained by induction at low temperature and purification.The prepared anti-HpaA polyclonal antibody had good specificity,which laid an experimental foundation of the development of H.pylori-related diagnostic reagents.

Crude extract of Periplaneta americana was prepared by liquid nitrogen grinding.After being purified with DEAE Sephadex A-50 ion exchange chromatography,the protein content of the extract was determined and the extract solution was prepared at gradient concentrations.The crude extract and purified allergen at different concentrations were dotted respectively on nitrocellulose(NC) membrane.Patient serum,bio-IgE,sa-HRP,luminal regents were added to the membrane.The chemiluminescence was displayed by exposing to X-film.The result revealed that the minimum protein content of crude Periplaneta americana extract detected by CLIA is 0.87?g/ml,with 90% accordance to skin test positive patients,and 100% accordance to those with negative skin test and ELISA detection.

Objective To clone the gene of arginine kinase (AK) from Periplaneta americana, produce its recombinant protein and investigate its allergenicity. Method The cDNA of AK was cloned using specific primers from the total RNA of P. americana. The cloned gene was inserted into pMD18-T vector and digested by BamHI and HindⅢ. The cDNA was sequenced and subcloned into pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant AK (rAK) was purified by metal (Ni2+) chelating affinity chromatography. Its allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay (ELISA). Result The cloned cDNA ORF sequence (Accession no. EU429466) contained 1 068 bp and encoded 365 amino acids. Its sequence homology with the published one (Accession no. AY563004) was 99.9% at nucleotide level. The allergen rAK was highly expressed in E. coli BL21 (DE3) as a soluble protein mainly with the molecular weight of about Mr 45 000 under induction of IPTG and purified by 6-His-tag purification system. Both in the non-denaturalization and denaturalization conditions, the recombinant allergen was identified as its affinity to IgE antibodies from the cockroach-allergic patient sera by Western blotting and ELISA. Conclusion The recombinant cockroach arginine kinase has been obtained with proper allergenicity.

Objective To clone,express and identify Der f 3 gene.Methods Live mites were collected from southern China region,identified as Dermatophagoides farinae,and cultured.The total RNA was extracted.The Der f 3 gene fragment was amplified by RT-PCR and sequenced.The Der f 3 gene fragment encoding a serine protease mature peptide was sub-cloned into the expression vector pET-His.The recombinant pET-Der f 3 plasmid was inserted into E.coli BL21 and induced to express Der f 3 coding protein by IPTG.The recombinant Der f 3 with 6 his-tag was then purified by chelating resin and its allergic activity was identified by Western blotting.Results The Der f 3 gene fragment with 840 bases was determined.Its sequence homology with the published one(GenBank No.D63858) was 99.5% at nucleotide level.It was sub-cloned into expressing vector pET-His and the recombinant allergen rDer f 3 was highly expressed in E.coli BL21(DE3) under induction of IPTG,and purified by 6-His-tag purification system.Using Western blotting method,the allergic activity of the purified recombinant allergen was identified as its affinity to IgE antibodies from the mite-allergic patient sera.Conclusion Der f 3 gene has been successfully cloned and its prokaryotic expression vector is constructed.

Since the study of the mechanism of breast cancer occurrence and development deepens, breast cancer stem cells are receiving more and more attention. Studies have shown that a group of breast cancer stem cells were undifferentiated, with self-renewal and multi-differentiation potential. These cells have a resistance to chemotherapy, radiotherapy, hypoxic, high tumorigenic, high invasion and metastasis. In breast cancer's recurrence,development, and even metastasis, they play an extremely important role. In-depth study of breast cancer stem cell related signal transduction pathways and the regulation of microenvironment are meaningful for clinical targeted treatment of breast cancer. Therefore, we summarized the latest development on breast cancer stem cells in the treatment of breast cancer.

Objective To investigate the treatment effect of aescuven forte on the postoperative complications of breast cancer.Methods One hundred and twenty patients with breast cancer radical surgery were randomly divided into control group(n =60) and treatment group(n =60).Patients in control group were given postoperative routine treatment,while in treatment group were administrated aescuven forte pills at 300 mg orally,2 times/day for 4 weeks beside the conventional treatment.Results (1)After the 1st,2nd weeks therapy,the flap congestion disappear rate in the treatment group were 80.0％ (48/60) and 93.3％ (56/60),better than that in the control group 60.0％ (36/60) and 71.6％ (43/60),the difference was statistically significant (P ＜ 0.05).However this trend was not seen in third weeks treatment (P ＞ 0.05).(2) The total efficiency of remission of upper limb edema was 93％ (56/60),higher than that in control group 77％ (46/60),the difference was statistically significant(x2 =5.17,P ＜ 0.05).(3) Visual analogue scale(VAS) pain score in treatment group were decreased form (8.87 ±0.74) in before treatment to (3.21 ±0.92) at after treatment.And the VAS score in control was from (8.91 ±0.85) down to (4.87 ± 1.34),the difference was statistically significant (P ＜ 0.05).Moreover VAS score in treatment group was lower than that in control group (P ＜ 0.05).(4) There was no adverse effect of the medication process.Conclusion Aescuven forte showed a ability to reduce congestion disappear time of breast cancer and shorten the recovery time of upper limb swelling and pain and other symptoms.

Objective To study the cotransfection mB7-1 and mCD1D gene into pancreatic cancer cells of rats and to observe its anti-tmor responses.Methods Recombinant retroviral vectors expressing mB7-1and mCD1D gene were packaged into GP2-293 cell lines and transfected.The expressions of mB7-1 and mCD1D were detected with PCR and Western blot.The positive cells of mB7-1 and mCD1D were used to induce the anti-tumor immunity in vitro.Results Anti-tumor immunity was induced after B7-1 and CD1D positive cells were coinoculated in syngeneic mice.Furthermore,the growth of tumor was inhibited.Conclusions Cotransfection of B7-1 and CD1D could induce anti-tumor effect.This study provide a foundation for the application of B7-1 and CD1D gene therapy in tumor.

This study aimed at investigating the effect of miR-21 on the apoptosis of hepatocellular carcinoma HepG2 cells induced by ursolic acid(UA). MTT assay was used to determine the inhibition effect of ursolic acid on proliferation of hepatocellular carcinoma cells. The expression level of miR-21 in hepatocellular carcinoma cells and the regulation effect of ursolic acid on the expression of miR-21 in HepG2 cells were determined by qPCR. To up-regulate the expression of miR-21, miR-21 mimics were transfected into HepG2 cells. Then MTT assay, flowcytometry(Annexin V-FITC staining), and RT-PCR were used to detect the regulation effects of ursolic acid on the proliferation, apoptosis, and the expression of apoptosis-related genes after miR-21 over-expression. The results showed that the proliferation inhibition effect of ursolic acid on HepG2 cells and the expression level of miR-21 in HepG2 cells were higher than in hepatic cell L-02 and hepatocellular carcinoma SMCC-7721, Bel-7402 cells. So further study was performed in the HepG2 cells. Ursolic acid inhibited the expression of miR-21 in HepG2 cells. And the greatest inhibition effect was at 24 h after treatment with UA. Over-expression of miR-21 partially offset the effects of ursolic acid on the proliferation, apoptosis, and the expression of apoptosis-related genes such as Bcl-2, survivin and Bax after 24 h. The results suggested that apoptosis of hepatocellular carcinoma HepG2 cells could be induced by ursolic acid by down-regulating the expression of miR-21.

Abstract: This study explores the potential antiepileptic mechanism of ursolic acid (UA) and its improvement of GABAergic interneuron damage induced by epilepsy based on transcriptome analysis. Hippocampal tissues from rats in the control group (NC group), epilepsy group (SE group), and epilepsy UA treatment group (UA group) were subjected to full-length transcriptome sequencing. The obtained sequencing data were analyzed, using gene ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) to perform the analysis of differential genes (DEGs). The expression levels of key differential genes were verified using RT-qPCR in hippocampal tissue. Finally, an epilepsy in vitro model was constructed on primary neurons, RT-qPCR was used to verify the expression levels of key differential genes, and the expression level of GABAA receptor γ2 subunit (GABRG2) on neurons was further examined using immunofluorescence and Western blot. The heatmap of pairwise sample expression correlation and the clustering analysis of differentially expressed genes showed that the SE group was farthest from the NC group, and that after UA treatment, the overall trend shifted towards the normal group. Compared with the SE group, a total of 220 differential genes were screened in the UA group, including 143 upregulated genes and 77 downregulated genes. GO enrichment analysis showed that it involved three processes in the primary classification: biological processes, cellular components, and molecular functions. KEGG pathway enrichment analysis showed that DEGs were involved in 36 biological pathways, including cAMP signaling pathway and calcium signaling pathway. PPI analysis showed that DEGs were closely related to GABA and inflammation. RT-qPCR results showed that UA treatment increased the expression levels of GABA receptor-related gene (Gng4), GABA synthesis-related gene (Camk2a,Vgf, and Npy) and inflammation-related gene (Timp1 and Spp1) in hippocampal tissue, and decreased the expression levels of GABA synthesis-related gene (Nptx2) and cAMP-related pathway gene (Gnas). It further confirmed that UA treatment increased the expression levels of Gng4 and Camk2a on neurons and decreased the expression level of Gnas. Immunofluorescence and Western blot results showed that, compared with the SE group, the expression level of GABRG2 on primary neurons increased after UA treatment. This study enriched the transcriptome data of UA's antiepileptic effect and laid a theoretical foundation for further research on UA's antiepileptic and neuroprotective effects.

Objective:To analyze the related factors affecting the dose to the small intestine exposure to preoperative radiotherapy for rectal cancer, aiming to provide reference for alleviating the adverse reactions of radiotherapy for rectal cancer.Methods:Medical record data and radiotherapy plan information of 138 rectal cancer patients who received intensity-modulated arc radiotherapy at Sun Yat-sen University Cancer Center from May 2021 to February 2023 were retrospectively analyzed. Tumor staging, tumor location, gender, age, planned bladder volume, body mass index (BMI), and small intestine irradiation dose volume were subject to Spearman correlation analysis. Further grouping and comparison were conducted based on the correlation results. Independent sample non parametric tests were used for inter group comparison.Results:The main factors related to the small intestine irradiation dose volume were tumor location, gender, planned bladder volume, and BMI. Tumor location was weakly correlated with the small intestine V 5 Gy-V 45 Gy. Gender was weakly correlated with the small intestine V 30 Gy-V 45 Gy. Planned bladder volume was weakly negatively correlated with the small intestine V 20 Gy-V 45 Gy. BMI was weakly negatively correlated with the small intestine V 10 Gy-V 45 Gy. Grouping comparison analysis showed that the small intestine V 5 Gy-V 45 Gy of rectal cancer patients in the low position group was significantly smaller than those in the middle and high position groups (both P<0.05), and there was no significant difference between the middle and high position groups ( P>0.05). Female rectal cancer patients had higher V 30 Gy-V 45 Gy levels than male counterparts ( P<0.05). The small intestine V 20 Gy and V 25 Gy levels in the planned bladder volume <200 ml group were significantly higher than those in the 200-400 ml and >400 ml groups (all P<0.05), whereas there was no difference between the 200-400 ml and >400 ml groups ( P>0.05). The small intestine V 30 Gy-V 45 Gy levels in the 200-400 ml group were significantly lower than those in the <200 ml group, but higher than those in the >400 ml group, and the differences were statistically significant (all P<0.05). Regarding BMI comparison among groups, the small intestine V 15 Gy-V 45 Gy in the low body weight group was significantly higher than those in the other three groups (all P<0.05). There were no significant differences among the normal, overweight, and obese groups (all P>0.05). Conclusion:In preoperative radiotherapy for rectal cancer, more attention should be paid to the dose to the small intestine in patients with middle and high position rectal cancer, female patients, and patients with low body weight.