Objective To evaluate the feasibility of transfection of Sonic hedgehog gene (SHH)into bone marrow mesenchymal stem cells(BMMSC).Methods After the SHH gene was transfected into BMMSC by electroporation apparatus,the transfection rate was evaluated by fluorescence inverted microscope.The growth curves of untransfected and transfected BMMSC were drawn,respectively,to observe the influence of transfection on cells.The expression of SHH gene in the BMMSC was detected by PCR,RT-PCR,Western-blot analyses.Results Through fluorescence inverted microscope,the observed transfection rate was appropriately 30％,PCR showed a obvious increase of SHH expression in transfected cells than that in untransfected cells,and it is quantified by qPCR for appropriately 7 times.Western-blot further demonstrated that the SHH protein expression in transfected cells had a distinct increase.However,it was observed that the exponential phase of BMMSCSHH growth curve delayed.The growth curves of both overlap 12 days after transfection.Conclusions This electroporation method can transfect exogenous SHH gene into BMMSC sufficiently with the effective protein expression in BMMSCSHH.It is the foundation of further research of genetic therapy for ischemic heart disease.

Objective　To investigate the expression of TGF-β and TGF-β receptor in human breast cancer cell Bcap-37 inhibited by soybean isoflavones. Methods　mRNA and protein of TGF-β1、TGF-βRⅠ in Bcap-37 cells were examined with reverse transcription ploymerase chain reaction(RT-PCR) and immunohistochemistry after cells were treated with daidein or genistein for 1-4 d.The expression of TGF-β1 and TGF-β2 was determined with TGF-β resistance test. Results　The TGF-β1, TGF-β2 and TGF-β recepor increased in Bcap-37 cells at a concentration of 3×10-5 mol/L of genistein. No changes was found when treated with daidzein. Conclusion　Genistein may inhibit the proliferation of Bcap-37 cells and accompany with increasing expression of TGF-β1, TGF-β2 and TGF-β receptor.

Aim To study the effect of tetramethylpyrazine(TMP) on binding of 125I-VEGF to VEGF receptor. Methods The mice sera were collected after peritoneal injection with big-dose TMP,low-dose TMP,protamine and NS. A reversed-phase high performance liquid chromatography(RP-HPLC) method was used to determine the TMP in mice serum. The culture medium of ECV304 was treated with the mice sera in different groups. Radioligand binding assay(RBA) of receptor and Scatchard pot were performed to observe the changes of the maximum binding capacity(B_ max) and dissociation constant(K_d).Results The sera of big-dose TMP inhibited 125I-VEGF binding to its receptor, K_d=343.30?36.64 pmol?L-1,B_ max=46.26?5.85 fmol/2?10~5 cells(P0.05),but B_ max decreased(P

AIM To evaluate the integration method for analysis of voltage-dependent Ca2+-independent transient outward K+ currents (Ito) in pharmacology. METHODSThe inactivation phases of Ito were best fitted by the sum of two or three exponentials equations. The area under the raw current curves (AUC) was obtained by the integration of exponential equations. The AUC normalized to the cell capacitance represented the net K+ charge flow during any depolarized duration and was as the index for comparison. Calcineurin overexpression transgenic (TG) mice showed downregulation of Ito. These data were tested by the integration method. RESULTS AUC obtained from three or two exponentials fittings was calculated as: AUC=A1τ1+A2τ2+A3τ3+A0t-A1τ1e-t/τ1-A2τ2e-t/τ2-A3τ3e-t/τ3 or AUC=A1τ1+A2τ2+A0t-A1τ1e-t/τ1-A2τ2e-t/τ2. The 50% and 90% action potential duration (APD50, APD90) in ventricular myocytes of mice are about 10 ms and 30 ms, respectively. AUC at 10 ms (AUC50, AUC of 50% APD) and 30 ms (AUC90, AUC of 90% APD) in left ventricle cardiomyocytes of wild type (WT) and TG mice were normalized to the cell capacitance. The normalized AUC50 and AUC90 of WT group were significantly more than those of TG group, which was consistent to the prolongation of APD in TG mice and the previous published results(downregulation of components of Ito in TG mice). CONCLUSION The integration method was an ideal way for analysis of transient outward K+ currents in pharmacology.

Objective To investigate the survival and differentiated situation of transplanted mesenchymal stem cells ( MSCs) transfected with Sonic hedgehog (SHH) gene after chronic myocardial infarction (MI).Methods MSCs were obtained from bone marrow of limb bones by density gradient centrifugation combined with attachment culture method .SHH gene was cloned and eukaryotic expression plasmid pcDNA 3.1-SHH was constructed .Then nucleofector TM was used to transfer SHH gene into MSCs and the expres-sions of mRNA and protein of SHH gene were detected in vitro .Myocardial infarction model was established by ligating the left anterior descending branch .A total of 150μl (3 ×106 ) of MSCs transferred with SHH gene was injected into ischemic area at the fourth week after MI model was set up .Brdu immunohistochemical staining and electron microscope examination were used to detect the survival and differentiation of MSCs with SHH in vivo at the 1st, 2nd, 4th, and 8th week after MSCs SHH transplantation.Results It was con-firmed that SHH gene clone and the recombinant eukaryotic expression vector pcDNA 3.1-SHH construction were successful by gene se-quence analysis and double digestions with EcoR I and Bamh I .Reverse transcriptase polymerase chain reaction ( RT-PCR) and West-ern-blot demonstrated that the expression of SHH mRNA was significantly increased in transfected MSCs compared with untransfected MSCs.Brdu immunohistochemical staining confirmed MSCs with SHH were survived from one to eight weeks in MI area after transplan -tation.Electron microscope examination showed that MSCs had the trend to be differentiated into myocardiac -like cells in MI area at the 4th and 8th week after transplantation .Conclusions The expression of SHH mRNA was significantly increased while MSCs were transfected with SHH gene , and MSCs SHH could survive and differentiate in good condition in the transplanted recipients .Those data provide the experimental basis for further transgenic cell therapy research of ischemic heart disease .

BACKGROUND AND OBJECTIVEStudies of passive smoking exposure in China however are of particular interest, because of the high lung cancer rate in people who are mostly non-smokers. The aim of this study is to explore the relationship between passive smoking and lung cancer among non-smoking Chinese.

METHODSBy searching Medline, PubMed, CENTRAL (the Cochrane central register of controlled trials), CBM, CNKI and VIP, et al, we collected both domestic and overseas published documents between 1987 and 2007 on passive smoking and lung cancer among non-smoking Chinese. Random or fixed effect models were applied to conduct meta-analysis on the case control study results, and the combined odds ratio (OR) and the 95% confidence interval (CI) were calculated as well.

RESULTSSixteen documents were included into the combined analysis, which indicated that there was statistical significance between passive smoking and lung cancer (OR = 1.13, 95% CI: 1.05-1.21, P = 0.001). It was significant of lung cancer among non-smoking subjects associated with amount of tobacco passively smoked more than 20 cigarettes daily, with life period in adulthood passive smoking exposure, with gender female, and with exposure to workplace. The P value, OR and 95% CI were P = 0.0003, OR = 1.78, 95% CI: 1.30-2.43; P = 0.0001, OR = 1.50, 95% CI: 1.23-1.83; P = 0.000 7, OR = 1.50, 95% CI: 1.19-1.90; P < 0.0001, OR = 1.41, 95% CI: 1.19-1.66; respectively. And there was no significant difference between passive smoking and lung cancer with amount of tobacco passively smoked within 20 cigarettes daily, with life period in childhood passive smoking exposure, with gender male and with exposure to spouse and parents.

CONCLUSIONPassive smoking is an important risk factor of lung cancer among non-smoking Chinese, and for non-smoking women who expose to environment tobacco smoke in a long period of time have a close relationship with lung cancer risk.

China ; epidemiology ; Female ; Humans ; Lung Neoplasms ; etiology ; Male ; Risk Factors ; Tobacco Smoke Pollution ; adverse effects ; statistics & numerical data

Objective:To analyze the medication rules in the ancient book Pu Ji Fang for the external treatment of acne based on data mining method. Methods:By screening out the methods of treating acne externally in Pu Ji Fang and establishing a standardized medical record database, this paper adopted the web version of Ancient and Modern Medical Record Cloud Platform to calculate the frequency, properties, flavors, and meridians of those medicines, and conduct cluster analysis by using IBM SPSS Modeler 18.0 software to analyze the association rules. Results:A total of 87 prescriptions were selected, including 164 kinds of Chinese materia medica, among which. Radix Angelicae, Ligusticum Wallichii, Rhizoma Typhoni and lead powder are frequently appeared. The properties of those medicines are mainly warm, cold and mild; the flavors of those medicines are mainly spicy, acrid, sweet and bitter, and the meridians mainly belongs to lung, spleen, stomach and liver meridians. The medical pair and group with the strongest associationion are Ligusticum Wallichii- Radix Angelicae and Rhizoma Typhonii- Radix Angelicae- Ligusticum Wallichii. Those freuently appeared medicines could be grouped into three categories. The paste dosage that was frequently appeared has strong correlation with tallow, mercury and lead powder, while the powder dosage that was frequenctly appeared has strong correlation with Angelica Dahurica, Radix Saponicae, Gleditsia sinensis, Radices Ligustici Sinensis and Ligusticum Wallichii. Conclusions:The application of data mining method could preliminarily reveal the medication rules of Pu Ji Fang for the external treatment of acne. The main treatment method is XinSanFaYue. The three categories of Chinese materia medica are used to treat the syndrome of asthenic habitus attacked by exogenous pathogenic factors, exterior attacked by wind heat and hot blood stasis respectively, showing the rules of treating acne externally before Ming Dynasty and providing references for the clinical treatment of acne.

Objective:To collect and analyze the evaluation index of infertility treated by Traditional Chinese Medicine (TCM), which will lay a foundation for the establishment of the core index and evaluation index system of TCM treating infertility.Methods:By retriving the published literatures of randomized controlled trials of infertility treated by TCM in the database of CNKI, VIP, Wanfang, SinoMed, PubMed and Cochrane Library from January 1, 2011 to December 31, 2020, and after the screening according to the creteria of inclusion and exclusion, this paper analyzed the normal indexes of infertility and the pathogenic indexes of TCM.Results:A total of 95 RCTs were included, including 9 069 patients aged between 20-39 years old. The average sample size of each RCT study was 95, involving 78 evaluation criteria. The highest frequency of use was the total effective rate, etiological-index analysis found that the general indicators of infertility such as the total effective rate, pregnancy rate, TCM syndrome scores,and common etiological factors such as LH, FSH are the characteristic indicators.Conclusion:There are many problems in the evaluation indexes of clinical trials of treating infertility by TCM, such as great differences in number and not standardized usage. It is necessary to carry out research on the construction of core indicator and evaluation indicator systems of infertility according to different etiologies.

Objective:To analyze the inflammatory mechanism and potential intervention drugs related to angiotensin converting enzyme 2 (ACE2) inhibitory mutations in order to provide reference for the treatment of corona virus disease 2019 (COVID-19).Methods:The data of lung adenocarcinoma with ACE2 mutations were screened from the cancer genome atlas (TCGA) database. The data were analyzed by R program language edgeR package and cluster Profiler package, gene ontology (GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Using String online analysis website for protein-protein interaction (PPI) network analysis, screening out the core genes, and finally using the Epigenomic Precision Medicine Prediction Platform (EpiMed) for multi-group association analysis of key genes, and drug candidates prediction.Results:A total of 1 005 differential genes were obtained, of which 91 were up-regulated and 914 down-regulated. A total of 71 GO were enriched, including 45 items related to biological processes, 16 items related to cell components, and 10 items related to molecular function. A total of 13 KEGG pathways were enriched, mainly in inflammatory pathways, various viral infectious diseases, transcriptional regulation, drug metabolism and protein digestion and absorption pathways. The differentially expressed genes were introduced into String online analysis website for PPI network analysis, a total of 252 proteins were obtained, and 10 core genes were H2A clustered histone 16(HIST1H2AL), H3 clustered histone 2 (HIST1H3B), H3 clustered histone 7 (HIST1H3F), H3 clustered histone 11 (HIST1H3I), H3 clustered histone 3 (HIST1H3C), H2B clustered histone 3 (HIST1H2BB), H2B clustered histone 6 (HIST1H2BI), H4 clustered histone 2 (HIST1H4B), H1-4 linker histone (HIST1H1E), H2A clustered histone 4 (HIST1H2AB). Interferon-α, resveratrol, celecoxib, heartleaf houttuynia herb, weeping forsythia capsule, dexamethasone, Chinese pulsatilla root, tumor necrosis factor-α inhibitors, liquorice root and famciclovir might be drugs for the treatment of ACE2 mutation-related inflammation.Conclusions:Inflammation associated with ACE2 inhibitory mutations is similar to the pathogenesis of COVID-19, which could lead to disease by promoting the activation of inflammatory pathways such as mitogen-activated protein kinase (MAPK), the Janus kinase signal transducer and activator of transcription (JAK/STAT), mammalian target of rapamycin (mTOR). Celecoxib, interferon and resveratrol may have the potential therapeutic effects on COVID-19.

Objective:To study the regulatory effect of miR-9 on the proliferation of breast cancer cells by targeting hexokinase 2 (HK2) .Methods:Breast cancer tissues and paracancer tissues were collected and the expression levels of miR-9 and HK2 were detected. MCF-7 cells were cultured and divided into blank control group, NC group, miR-9 group, NC-siRNA group and HK2-siRNA group. The cell viability, expression levels of HK2 and cleaved caspase-3 were detected, the targeted binding of miR-9 to HK2 was verifed.Results:the expression level of miR-9 in breast cancer was lower than that in paracancer tissue (0.52±0.08 vs 1.05±0.25, t=16.685, P<0.000) , and the expression level of HK2 was higher than that in paracancer tissue (0.73±0.14 vs 0.34±0.08, t=17.587, P<0.000) , and the expression level of miR-9 was negatively correlated with HK2; the OD490 level, the expression level of HK2 and the fluorescence activity of double luciferase reporter gene containing HK2 mRNA 3 'UTR in miR-9 group were lower than those in NC group (0.58±0.09 vs 1.04±0.21, 0.51±0.08 vs 1.18±0.24, 41.11±9.28 vs 148.28±29.59, t/P=4.027/0.007, 5.297/0.002, 6.912/0.001) , and the expression level of cleaved caspase-3 was higher than that in NC group (1.08±0.26 vs 0.42±0.09, t/P=4.797/0.003) . The OD490 level and the expression level of HK in HK-siRNA group were higher than that in NC-siRNA group, and the expression level of cleaved caspase-3 was higher than that of NC-siRNA group. Conclusion:miR-9 can inhibit the proliferation of breast cancer cells, and its mechanism may be related to the targeted inhibition of HK2 expression and the increase of downstream cleaved caspase-3 expression.