Objective To study whether RHD gene exists multiple mRNA produced by alternative splicing. Method RhD mRNA was analyzed through reverse transcriptase PCR (RT-PCR) and cDNA sequencing in 4 individuals with different Rh phenotypes (CcDDEe、CCDDee、CcDDee and ccDdee). Results The individuals with different Rh phenotypes had identical and intricate RhD mRNA isolations, which included normal mRNA, and 4 other transcripts with deletions of exon 7, exon 9, exon 7 and 9, and exon 7 to 9. Those transcripts had the same sequences as normal RhD mRNA except exon(s) deletion, which showed RHD gene existed alternative splicing, and it happened in exons or introns 7-9 regions. Conclusion There are intricate and different cassette-exons RhD transcripts produced through alternatively spliced exons 7 to 9. And those RhD mRNA isolations might code proteins with different C-terminus.

Objective To establish a RHD genotyping method specific for the Chinese. Methods Six pairs of primers specific for most alleles found in the Chinese according to the records in NCBI GenBank, were designed, and a multi-tube sequence-specific primer PCR (PCR-SSP) method was established with a pair of internal control primer in each reaction. The method was evaluated with samples serologically determined and full length RHD sequenced from 89 Rh-negative, 28 D el, and 13 Rh-positive, weak D and partial D phenotype of Chinese Hans. Furthermore, 318 random samples from blood donors were genotyped and the results were compared with serological results of those samples. Results The PCR-SSP results were in concordance with serological results (100%) in all samples, and all RHD positive, D antigen negative alleles (or nonfunctional alleles) observed in the Chinese up to now could be detected or implicated, including D el phenotype especially D el allele existing in Rh-positive individuals (RHD/RHD1227A). This genotype was detected with a rate of 8/318, and allele frequency should be 0.012579 Conclusion Our method is rapid and easy, with high accuracy in the testing of the Chinese.

Objective To establish fully automated sample pooling, nucleic acid extraction, amplification and detection method for HBV DNA testing, and investigate the seroconversion and genotype in HBV DNA positive donors. Methods Individual donor plasma samples serologically negative for HBV were pooled by STAR2000 sampling processor with a size of 24. Nucleic acid were automatically extracted by MPLC simultaneously, amplified and detected by Roche COBAS AMPLICOR system. The sensitivity of detection was determined by international standard. HBV DNA positive donors were genotyped and followed up by serological tests. Results The 95% detection limit for this automated HBV DNA testing system was 38.9IU/ml,with 95% confidence interval (21323),eight out of 16512 specimens were PCR positive for HBV DNA,with a positive rate of 0.049%. Three of the 8 DNA positive donors were genotype C,2 genotype B, 1 genotype D,and the other 2 uncertain。Six of the eight HBV DNA positive donors were followed up, and three of them seroconverted。 Conclusion Fully automated HBV DNA detection method can be applied in blood screening,and will further increase the safety of blood supply.

BACKGROUND:It is a great potential study that calcium sulfate product loaded with antibiotics is developed, but this product is not systematicaly studied and its biocompatibility and security need to be further studied.
OBJECTIVE:To evaluate the biocompatibility and safety of vancomycin- or gentamicin-loaded calcium sulfate bone.
METHODS: (1) Hemolysis test: vancomycin-loaded, gentamicin-loaded calcium sulfate extracts, double distiled water and saline were added into rabbit anticoagulant blood samples. (2) Micronucleus test: vancomycin-loaded and gentamicin-loaded calcium sulfate extracts, cyclophosphamide and normal saline solution were intraperitonealy injected to mice, respectively. (3) Acute toxicity test: vancomycin-loaded and gentamicin-loaded calcium sulfate extracts, and normal saline solution were intraperitonealy injected to mice, respectively. (4) Pyrogen test: the mice were injected vancomycin-loaded and gentamicin-loaded calcium sulfate extractsvia the ear vein. (5) Intradermal stimulation test: vancomycin-loaded and gentamicin-loaded calcium sulfate extracts were respectively injected into the unilateral spine of rabbits, respectively. (6) Intramuscular implantation test: vancomycin-loaded and gentamicin-loaded calcium sulfate extracts were respectively injected to the dorsal muscle of rabbits. (7) Intraosseous implantation test: vancomycin-loaded and gentamicin-loaded calcium sulfate were implanted into the necrotic femoral bone of rabbits.
RESULTS AND CONCLUSION:Both vancomycin-loaded and gentamicin-loaded calcium sulfate products, which have no hemolytic reaction, genetic toxicity, acute toxicity, pyrogen reaction and skin irritation, are considered to have good biocompatibility and safety.

Objective To study an improved isolated method of single human atrial myocytes.Methods Enzyme digestion method was used to isolate single myocytes from human atrial and whole-cell patch clamp technique was used to record small conductance calcium activated potassium current.Results This method obtained a large number of atrial myocytes.The total amount of atrial myocytes in SR group was 320±30 while AF group was 230±20 and the difference was statistically significant(P<0.01).In this study,a large number of simple and striated single atrial myocytes were obtained,and a typical small-conductance calcium-activated potassium channel current was recorded on the isolated atrial myocytes.Conclusion The established isolated method is simple,stable and effective.We can acquire a large amount of single atrial myocytes with good quality.

Objective To investigate effect of early enteral nutrition supplemented with probiotics on gastrointestinal motility disturbance and nutritional status in patients with severe craniocerebral trauma.Methods Forty patients with severe craniocerebral trauma were randomized into study group (18patients) and control group (22 patients).Patients of both groups received enteral nutrition via nasogastric tube at 24-72 hours after admission,but the patients of study group were also supplemented with probiotics simultaneously.Rate of abdominal distention,vomiting,gastro-oesophageal reflux,gastric retention,constipation and diarrhea were recorded during the whole study.Time to first defecation and time to targeted nutritional goals were also recorded.Prealbumin and transferrin in serum were detected at days 0,4,7,and 15 after the beginning of enteral nutrition.Length of ICU stay was compared between groups.Results There were no significant differences of the two groups in terms of rate of abdominal distention,vomiting,gastric retention and diarrhea.However,less gastro-oesophageal reflux or constipation patients were observed in study group,as compared with control group (P ＜ 0.05).Time to first defecation and time to targeted nutritional goals were shorter in study group,as compared with control group (P ＜ 0.05).Levels of prealbumin and transferrin had no significant differences between the two groups at days 0,4,and 7,but study group showed both were higher than control group at day 15 (P＜0.05).Moreover,length of ICU stay showed no significant difference between the two groups.Conclusions Compared with simple enteral nutrition,early enteral nutrition with probiotics improves gastrointestinal motility,facilitates the delivery of enteral nutrition,and further ameliorates nutritional status in patients with severe craniocerebral trauma.

Objective To establish the entirely automatic method of nucleic acid amplification testing (NAT) for blood screening and to study the feasibility of NAT.Methods Using entirely automated extraction method to extract nucleic acid , amplified and detected by Roche COBAS AMPLICOR system,evaluating the sensitivity and efficacy.Results The 95% limits of HBV DNA, HCV RNA/HIV-1 RNA tests by automation system were 38.9,16.4IU/ml and 20.4 copies/ml,95% Confidence Intervals were [21,323], [10.5,342] and [12,300] respectively.8 of 16 512 donations were PCR for HBV DNA positive,the DNA positive rate was 0.048%.7/8 donations were Anti-HBc positive,The last one was also converted positive.No positive HCV RNA and HIV RNA was detected. 3/6 following up samples seroconverted.Conclusions The entirely automatic system can be applied in blood screening.

Objective:To investigate the correlation between variations in mitochondrial DNA (mtDNA) control region (D-loop) and keloids.Methods:A total of 216 patients with keloids were collected from Department of Dermatology, the First Affiliated Hospital of Kunming Medical University from 2016 to 2019. Total DNA was extracted from peripheral blood samples of all the patients, as well as keloid tissues and perilesional normal skin tissues of 25 patients with keloids. Peripheral blood samples were collected from 299 health checkup examinees without keloids in Health Examination Center, the Affiliated Hospital of Yunnan University, who served as controls. PCR amplification and Sanger sequencing were performed on the mtDNA D-loop region, and mutation sites in each sample were analyzed by comparisons with the revised Cambridge Reference Sequence (rCRS) . Haplogroups were assigned in the 2 groups by using Phylotree-mtDNA tree Build 17. Mutations in the mtDNA D-loop region were compared among keloid tissues, perilesional normal skin tissues and peripheral blood samples. A median-joining network was constructed via network 5.0 software. Binary logistic regression analysis was performed to investigate the correlation between haplogroup frequencies and the occurrence of keloids, and chi-square, t and t′ tests were used to analyze clinical data. Results:Among the 216 patients with keloids, variations in mtDNA D-loop region were classified into 10 haplogroups, including A, B, D, R9, G, M*, M7, M8, M9 and N9, with the haplogroups R9 and M9 showing the highest (21.3%, 46/216) and lowest (0.9%, 2/216) frequencies respectively. The frequencies of haplogroups M7 ( P=0.040, OR=0.248, 95% CI: 0.066 - 0.937) and N9 ( P=0.048, OR=0.191, 95% CI: 0.037-0.986) were significantly lower in the patients with keloids than in the controls. The median-joining network plot showed that the distribution pattern of the haplogroup M7 differed between the patients with keloids and controls. Significantly less number of lesional sites and younger age of onset were observed in the patients with haplogroup M7 compared with those with non-M7 haplogroups ( P=0.000 1, 0.045, respectively) . Conclusion:The haplogroup M7 is correlated with the occurrence of keloids, and may be a potential protective factor for keloid formation.