Objective To screen the Doc 1R gene recombinant plasmid by use of colony PCR. Method The recombinant colonies were transfered into the PCR reaction mixture. The PCR primers were used for constructing mouse Doc 1R genomic sequence. Result Among the 5, 3 positive strips in the size of 1 500 bp were visible, which were the same as the Doc 1R gene fractions in terms of their sizes were screened as positive clones. The positive colony were further confirmed by double digestion and DNA sequencing. Conclusion Colony PCR is a simple, efficient and reliable technique for screening the recombinant.

BACKGROUND: Establishment of drug (neomycin or hygromycin) resistant feeder layer cells is necessary for screening the target gene positive clone of embryonic stem cell (ESC) transfected in vitro, and the establishment of drug-resistant NIH3T3 cell line is also important for the screening of other target ESC genes.OBJECTIVE: To obtain a feeder layer of positive cell clone of ESC transfected by pTet-on gene by means of the G418-resistant NIH3T3 cell line establishment.DESIGN: Cell culture and DNA examination.SETTING: Faculty of Laboratory Animal, China Medical University.MATERIALS: NIH3T3 cells were contributed by the Cell Biology Staff Room of China Medical University, and pWL/neo plasmid was a gift from Professor Jin Zhuang of Harvard University Medical College. G418,leukemia inhibitory factor (LIF, ESC GRO 106 U/mL) and DMEM were produced by GIBCO BRL Company, while mitomycin-C (MIT-C), DIG marking and kits were the products of Roche Company. Lipoetin was the product of Invitrogen Company, and bought from Shenyang Lianxing Biotechnology Co.,Ltd.METHODS: ①The pWL/neo plasmid containing neo gene was purified and transfected into NIH3T3 cells by lipoetin.②The transfected cells were further cultured in 25 mL medium containing G418 antibiotic of gradient concentration to observe the survival and apoptosis of cells and measure G418 minimal fatal dose (MFD) to NIH3T3 cells.③The transfected cells were subcultured, and the clone of single cell was selected to 24-pore culture board for screening amplification. At the same time, normal NIH3T3 cells were also selected and added with the selective culture medium at the same dose of G418, as screening negative control. ④The cells were planked with lower cell density, and further screened in DMEM medium containing G418 MFD; Meanwhile, normal NIH3T3 cells were taken as controls, and cultured by G418 contain MFD and by DMEM medium without G418 respectively. Light microscope was used to observe G418R NIH3T3 cells. ⑤G418R NIH3T3 cells and MEF were all applied as the feeder layer to culture the ES-D3 cells, which were observed by light microscope. Cell DNA was prepared, and evaluated by PCR and southern blot.MAIN OUTCOME MEASURES: ①G418 MFD to NIH3T3 cells. ②screening result of G418R NIH3T3 cells. ③growth of G418R NIH3T3 cells. ④growth of ES-D3 cells on G418R NIH3T3 cells.RESULTS: ①G418 MFD to NIH3T3 cells was defined as 500 mg/L.②Under the condition of G418 (500 mg/L), G418R NIH3T3 cell clones were successfully selected. ③The G418R NIH3T3 cells had no difference from normal NIH3T3 cells in morphology and propagation rate.④The ESC cultured in feeder layer present a colony growth, smooth limitation and remained an undifferentiation state. The resistant cell genomic DNA of G418R NIH3T3 cells was assayed with specific neo gene primer, and then neo gene DNA fragment was amplified; Southern blot analysis showed that neo gene fragment integrated into G418R NIH3T3 cells.CONCLUSION: The G418R NIH3T3 cells are established successfully,and the ESC cultured in the G418R NIH3T3 cell feeder layer can keep the characteristics of normal ESC.

Chinese traditional patent medicine for promoting blood circulation and removing blood stasis(PBCRBS) originated from traditional Chinese medicine theory and had approved efficacy and safety standards. However, its compatibility regularity and anti-thrombotic mechanism is not clear. To analyze the compatibility regularity and anti-thrombotic mechanism of Chinese traditional patent medicine for PBCRBS, a statistical and bioinformatics analysis was carried out using traditional Chinese medicine inheritance support system (TICMISS, V2.0) and ingenuity pathway analysis (IPA). The compatibility regularity analysis shows that the most commonly used herb combinations are Danshen (Salvia miltiorrhiza Bge.), Chuanxiong (Ligusticum chuanxiong Hort.) and Honghua (Carthamustinctorius L.). The anti-thrombotic mechanism analysis reveals that 25 ingredients have an effect on 29 thrombosis related molecules which 23 molecules are related to inflammation response. Furthermore, there are 5 inflammation molecules (NOS2, PTGS2, IL6, TNF, IL1β) served as major targets. At the same time, Danshen, Chuangxiong and Honghua mainly used as sovereign herb or minister herb in the application of cardiovascular and cerebrovascular diseases. Therefore, Chinese traditional patent medicine for PBCRBS probably has an effect on anti-thrombotic activity through inhibiting the inflammatory response. In summary, the most commonly used herb combinations of Chinese traditional patent medicine for PBCRBS are Danshen, Chuanxiong and Honghua. Inhibiting inflammatory response, especially inflammation related molecules (NOS2, PTGS2, IL6, TNF and IL1β), is probably a new starting point to clarify the anti-thrombotic mechanism of Chinese patent medicine for PBCRBS.

AIM To investigate the effect of Icarrin on penile erection and cyclic GMP levels in rabbit corpus cavernosum(RCC). METHODS Effects of various concentration of Icariin on cGMP level in RCC were measured by 125 I radioimmunoassay procedure with Sildenafil and papaverine as the positive controls RESULTS In the presence of SNP, Icariin increased cGMP concentration in rabbit corpus cavernosum in a dose dependent manner ( P

Objective: To search for specific metabolites in the lungs of pneumonia rats fed with a high-calorie diet, as well as explore the changes in the lung metabolites of young rats treated with Yinlai Decoction (YD) and its effects on inflammation-related metabolic pathways.Methods: Lipopolysaccharides (LPS) and a special high-calorie diet were used to induce Sprague Dawley (SD) rats to simulate the intestinal state of infant pneumonia. Liquid chromatography-mass spectrometry technology (LC-MS/MS) was used to detect metabolites in each group. Supervised orthogonal partial least squares discriminant analysis (OPLS-DA) model values were used for the detection results to find the differential metabolites. The metabolic pathways that are involved with the differential metabolites were clarified through enrichment analysis and topological analysis. Finally, the T cell receptor signaling pathway (TCR) signal conversion was analyzed by the network pharmacology method. Results: In the high-calorie diet combined with pneumonia group (M3), a total of 55 metabolites were determined to be different from the normal group (N). A total of 36 metabolites were determined to be different from those in the lung metabolites of the YD treatment group (T1). YD had a regulatory effect on glutathione metabolism, arginine and proline metabolism, ascorbic acid and aldehyde metabolism and phenylalanine metabolism. And the small molecule metabolites could act on the FYN and lymphocyte-specific protein tyrosine kinase (LCK) target proteins in the TCR signaling pathway, thereby affecting the immune function of the lungs. Conclusion: A high-calorie diet can cause abnormal sphingolipid metabolism in the lungs of young rats, thereby creating chronic lung inflammation in young rats. YD has a beneficial effect when used to treat young rats with LPS-induced pneumonia fed on high-calorie diets. Its mechanisms of action may affect the body's immune pathways by regulating the oxidative stress pathway affected by glutathione metabolism.

Background::Ainuovirine (ANV) is a new generation of non-nucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus (HIV) type 1 infection. This study aimed to evaluate the population pharmacokinetic (PopPK) profile and exposure-response relationship of ANV among people living with HIV.Methods::Plasma concentration-time data from phase 1 and phase 3 clinical trials of ANV were pooled for developing the PopPK model. Exposure estimates obtained from the final model were used in exposure-response analysis for virologic responses and safety responses.Results::ANV exhibited a nonlinear pharmacokinetic profile, which was best described by a two-compartment model with first-order elimination. There were no significant covariates correlated to the pharmacokinetic parameters of ANV. The PopPK parameter estimate (relative standard error [%]) for clearance adjusted for bioavailability (CL/F) was 6.46 (15.00) L/h, and the clearance of ANV increased after multiple doses. The exposure-response model revealed no significant correlation between the virologic response (HIV-RNA <50 copies/mL) at 48 weeks and the exposure, but the incidence of adverse events increased with the increasing exposure ( P value of steady-state trough concentration and area under the steady-state curve were 0.0177 and 0.0141, respectively). Conclusions::Our PopPK model supported ANV 150 mg once daily as the recommended dose for people living with HIV, requiring no dose adjustment for the studied factors. Optimization of ANV dose may be warranted in clinical practice due to an increasing trend in adverse reactions with increasing exposure.Trial registration::Chinese Clinical Trial Registry https://www.chictr.org.cn (Nos. ChiCTR1800018022 and ChiCTR1800019041).

COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread across the globe, posing an enormous threat to public health and safety. Traditional Chinese medicine (TCM), in combination with Western medicine (WM), has made important and lasting contributions in the battle against COVID-19. In this review, updated clinical effects and potential mechanisms of TCM, presented in newly recognized three distinct phases of the disease, are summarized and discussed. By integrating the available clinical and preclinical evidence, the efficacies and underlying mechanisms of TCM on COVID-19, including the highly recommended three Chinese patent medicines and three Chinese medicine formulas, are described in a panorama. We hope that this comprehensive review not only provides a reference for health care professionals and the public to recognize the significant contributions of TCM for COVID-19, but also serves as an evidence-based in-depth summary and analysis to facilitate understanding the true scientific value of TCM.