Establishment of PCR Identification Method for Pig Blood Type / 实验动物与比较医学

ObjectiveXenotransplantation is an effective way to address the shortage of human organ donors, but it faces serious immune rejection reactions, including hyperacute rejection caused by blood type differences. Establishing a stable, convenient, and reliable method for pig blood type identification can quickly screen suitable donor pigs for xenograft research.MethodsBanna miniature inbred pigs, Diannan small eared pigs, and Bama Xiang pigs were selected as the research objects. DNA was extracted from the blood, oral buccal mucosa, and fetal fibroblasts of the three strains of pigs using DNA extraction kits. The target fragment of the ABO homologous gene EAA intron 7 in pigs was amplified using PCR method. Blood agglutination reaction was used to detect hemolysis in pig anterior vena cava whole blood after adding anti A and B antibodies. Immunohistochemical method was used to detect the expression level of A antigen in pig heart, liver, spleen, lung, and kidney tissues. Immunofluorescence method was used to detect the expression level of A antigen in pig oral mucosa. By comparing the results of different methods for determining pig blood types, the stability and reliability of the PCR method were verified, and a convenient PCR based pigblood type identification method was established.Results Firstly, the blood PCR results of 69 inbred strains of Banna miniature pigs, 7 Diannan small eared pigs, and 34 Bama Xiang pigs showed 20 AO blood types, 66 AA blood types, and 24 O blood types. The PCR results of fetal fibroblasts from 47 Diannan small eared pigs showed that all 47 fetuses were O blood type. Among them, the oral mucosal PCR results of 8 gene edited cloned pigs were consistent with those of donor fetal fibroblasts, all of which were O blood type. The oral mucosal PCR results of 8 wild-type pigs (2 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs) were consistent with the blood PCR identification results. Then, 11 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs were randomly selected for blood agglutination reaction validation, and the results were consistent with the PCR identification results of both blood samples and oral mucosa samples. Moreover, immuno-histochemical analysis was performed on the heart, liver, lung, kidney, and spleen tissues of one Banna miniature pig inbred line and two Bama Xiang pigs, and the results were consistent with blood PCR identification and blood agglutination reaction results. Finally, oral mucosal samples were collected from 2 inbred strains of Banna miniature pigs and 1 Bama Xiang pig for immunofluorescence detection, and the results were consistent with the blood PCR identification results.ConclusionBy collecting fetal cells and oral mucosal samples from live pigs for PCR detection, the blood type of pigs can be accurately and efficiently identified, providing a convenient method for blood type screening of xenograft donor pigs.

Exploration and reflection on the online teaching of physiology under the COVID-19 epidemic / 中华医学教育探索杂志

Facing the challenge of the COVID-19 epidemic to the classroom teaching, the physiology teaching team of Qiqihar Medical University constructed a blended teaching model based on "small private online course (SPOC)+ live real audio" to carry out online teaching. Through the joint efforts of all team members, the online teaching has been carried out in a stable order for 4 weeks, thus ensuring the teaching effect of physiology. Taking the physiology teaching of 236 nursing undergraduates in Batch 2019 as an example, this paper introduces the teaching design, implementation measures, teaching effect and teaching reflection of carrying out online teaching under the epidemic situation, and provides practical experience for further promoting online teaching in medical colleges and universities during epidemic prevention and control.

Induction of thymidine phosphorylase expression by AZT contributes to enhancement of 5'-DFUR cytotoxicity.

Thymidine phosphorylase (TP) regulates intracellular thymidine metabolism and can enhance the anti-tumor effectiveness of 5'-deoxy-5-fluorouridine (5'-DFUR) by conversion of the pro-drug 5'-DFUR to 5-fluorouracil (5-FU) in tumor tissues. 5'-DFUR is an effective anti-tumor drug in cells expressing high levels of TP. 3'-Azido 3'-deoxythymidine (AZT) is a thymidine analog that has been proven useful in the treatment of acquired immunodefiency syndrome (AIDS). In this study, we found that AZT induces TP expression and enhances the sensitivity of human myeloid leukemia U937 cells to 5'-DFUR. Both the protein level and the activity of TP in U937 cells were elevated for 48h after exposure to AZT (20, 100 or 300muM). AZT enhanced TP promoter activity in a dose-dependent manner. AZT also increased TP mRNA levels in U937 cells as assayed by Real-time reverse-transcription PCR. AZT enhanced the cytotoxic effect of 5'-DFUR on U937 cells. A TP inhibitor, TPI, abrogated the cytotoxic activity of 5'-DFUR, and attenuated the combined cytotoxicity of AZT and 5'-DFUR. These results suggest that AZT enhances the cytotoxic effect of 5'-DFUR on U937 cells by upregulating TP activity in addition to its inhibition of thymidine kinase (TK) activity and reduction of intracellular dTTP pools.

Integration of quality education into physiology teaching reform / 中华医学教育探索杂志

According to the requirements of the ‘three guidance’ talent-cultivation model pro-posed by our university, humanistic spirit was permeated from multi angels during the process of phys-iology. Students'occupation quality was cultivated; physiology knowledge and clinical practice was combined for students; early contacting with clinical knowledge was conducted and teacher-student role reversal method was applied to train students' communication skills. Teaching practice showed that the reformed teaching model not only can ensure the quality of teaching but also can improve the comprehensive quality of students.

Preparation of proteins from different organs of Japanese morning glory by an in vivo electro-elution procedure.

An electro-elution procedure has been employed efficiently to collect proteins from stem segments, young leaves and roots of the Japanese morning glory. Electrophoretic conditions were optimised, including the size of segments (10-30 mm), the strength of the current for electro-elution (2.5-10 mA), and the exposure time of electro-elution (2-12 h). From the same quantity of organs, the in vivo electro-elution procedure permitted the collection of an amount of protein up to six times greater than that obtained with an earlier-reported centrifugation procedure. Both preparations were analysed by SDS-PAGE and showed similar protein profiles. This new technique provided an interesting insight into the large differences in both the quality and quantity of proteins between different organs of the plants. The average amount of protein collected from organs was 0.1 mg/g of tissue fresh weight. It is expected that this procedure may facilitate the discovery of new proteins with unique functions in extracellular matrices involved in the response of plants to various external stimuli.

Genetic analysis of ?-ADDUCIN and GNB3 in essential hypertension patient / 基础医学与临床

Objective To explore the polymorphism at position G460W of ?-ADDUCIN and at position C825T of GNB3,and the genetic interaction between ?-ADDUCIN and GNB3 genes in a QiQihr essential hypetension population.Methods Three hundreds and thirty-one patients with EH and two hundreds and ninety-three healthy controls were enrolled.Genotyping was performed using PCR-RFLP technique.Results(1)genotype distributions of ?-ADDUCIN G460W(GG 0.177 vs 0.160,GW 0.580 vs 0.481,WW 0.242 vs 0.359,P=0.006) and GNB3(CC0.177 vs 0.353,CT 0.468 vs 0.541,TT 0.355 vs 0.106,P