[Objective] To investigate the factors influencing the detection of HLA-C expression by flow cytometry. [Methods] A total of 12 hematopoietic stem cell suspension samples from peripheral hematopoietic stem cell volunteer donors were randomly collected after CD34⁺ cell counting detection. The influence of detecting different number of nucleated cell (500 000, 50 000 and 5 000), sequential order of red blood cell lysis and antibody incubation, and the HLA-C antibody with varied remaining time from the expiration date on the detection results of HLA-C expression by flow cytometry were investigated, respectively. The significance of differences between different groups was analyzed through Student t test. [Results] There was no significant difference in the proportion of HLA-C positive cells and mean fluorescence intensity (MFI) among the three groups with different nucleated cell numbers detected (500 000, 50 000 and 5 000) (P>0.05). The sequential order of red blood cell lysis and antibody incubation had no influence on the proportion of HLA-C positive cells (P>0.05), but HLA-C MFI value was significantly lower when antibody incubation was performed after red blood cell lysis than that when antibody incubation was performed before red blood cell lysis (P<0.05). The proportion of HLA-C positive cells and MFI value detected by HLA-C antibody remaining 24 months from the expiration date were significantly higher than those detected by HLA-C antibody remaining only 5 months from the expiration date (P<0.05). [Conclusion] The present study has investigated the factors of influencing HLA-C expression level by flow cytometry, the results have important reference and application value for standardizing the experimental operation of HLA-C expression and improving the accuracy and comparability of detection results.

Objective To generate minichromosome maintenance protein 2(MCM2)gene knockout cervi-cal cancer HeLa cell lines using inducible CRISPR/Cas9 technology,and to explore the effect of MCM2 on DNA replication and replication stress.Methods The inducible CRISPR/Cas9 system,TLCV2,was used to construct MCM2 knockout HeLa cell lines.And the cell lines were divided into control group(Control),knockout group 1(KO1),and knockout group 2(KO2).Western blot,Edu incorporation experiment,real-time quantitative PCR(qPCR),immunofluorescence and MTT assay were used to analyze the effects of MCM2 knockout on DNA replica-tion and replication stress induced by hydroxyurea.Results The CRISPR/Cas9 system successfully knocked out the MCM2 gene after induction,and MCM2 knockout affected the stability of MCM2-7 complex.Compared with the control cells,MCM2 knockout cells had a dramatic decrease in the capacity of DNA replication,and the mRNA levels of Cyclin A1,Cyclin E1 and CDK4.Under DNA replication stress,MCM2 knockout cells decreased cell viability,DNA damage repair capacity,and increased genomic instability compared with control cells.Conclusion Knockout of MCM2 gene reduces the DNA replication capacity of HeLa cells under normal conditions and cell viability under replication stress.This study successfully generates MCM2 gene inducible knockout HeLa cell lines,laying the foundation for further research on the role and biological function of MCM2 gene in the occurrence and progression of cervical cancer.

Objective To investigate the effect of the petroleum ether extract of Sageretia thea on the proliferation and apoptosis of breast cancer cells.Methods After breast cancer cells were incubated with the petroleum ether extract for different times,cell viability was analyzed by CCK8 assay,cell proliferation was detected by plate cloning test,nuclear morphology was observed by DAPI staining,mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)were determined by immunofluorescence,and the cell cycle and apoptosis were detected by flow cytometry.After incubating with the extract for 24 h,the CCK8 assay was used to observe the toxicity to normal human vascular endothelial cells.Results The IC50 of BT549 and MDA-MB-231 breast cancer cell lines treated with the petroleum ether extract for 24 h were 45.40 μg/ml and 12.23 μg/ml,respectively.The extract time and dose dependently inhibited breast cancer cell viability and clonal formation,induced cell apoptosis and cycle arrest in G1/S phase,decreased MMP and increased ROS levels.There was no toxic effect on normal endothelial cells.Conclusion The petroleum ether extract of Sageretia thea may induce apoptosis by increasing ROS to cause MMP collapse,followed by activating mitochondrial pathway,thereby hindering the growth of breast cancer cells.These results could support the application of Sageretia thea to anti-breast tumor in the folk.

Objective To establish a method for qualitative detection of the presence or absence of all KIR genes by quantitative polymerase chain reaction(Q-PCR).Methods Based on the polymorphism of high-resolution level KIR alleles in Chinese population and the IPD-KIR database,KIR gene-specific primers were designed to amplify all the 16 KIR genes and 2DS4-Normal and 2DS4-Deleted subtypes by Q-PCR.Meanwhile,one negative control and one positive control specific amplifying human growth hormone(HGH)gene fragment were set to monitor the false positive and false negative results in PCR amplification,respectively.A total of 302 samples with known KIR genotype previously identified by KIR PCR-SSP commercial kit were randomly selected for blind inspection to verify the reliability of KIR Q-PCR method established by au-thors.Results The results of 300 samples detected by our KIR Q-PCR method were consistent with the known results,but two samples showed inconsistent results.One sample was negative for 2DS5 by Q-PCR but positive by PCR-SSP,another sample was positive for 2DS1 by Q-PCR but negative by PCR-SSP.The two doubtful samples were genotyped by sequencing-based typing(PCR-SBT)for 2DS5and2DS1,respectively.PCR-SBT results confirmed that the results of Q-PCR test was correct.Conclusion The KIR Q-PCR method established in this paper can provide accurate and reliable results for testing the presence or absence of KIR genes.

Objective:To study the genetic polymorphisms of short-tandem repeats (STR) for the D13S317 locus among an ethnic Han Chinese population and verify a novel tri-allelic pattern identified for the locus. Methods:A total of 378 paternity test cases from Guangdong Forensic Authentication Institute from October 17, 2017 to December 28, 2017 were selected as the study subjects. A GlobalFiler? Express kit was used for the STR genotyping. Samples suspected for having a novel tri-allelic pattern were verified with a PowerPlex ? 21 kit. Potential variant of the primer-binding region and flanking sequences underlying the tri-allelic pattern was excluded by molecular cloning and sequencing. Results:Six alleles were detected for the D13S317 locus, with the characteristic distribution frequencies being 8 (29.1%), 9 (13.1%), 10 (15.21%), 11 (24.21%), 12 (13.89%) and 13 (3.44%), respectively. In one of the families, the D13S317 locus of the proband was suspected to harbor a triband allele (8, 9, 10). A re-test has confirmed the result of initial test. Molecular cloning and sequencing analysis of the D13S317 locus in the proband and his daughter has failed to find allelic variants in the primer-binding region and flanking sequence, which has confirmed the novel tri-allelic pattern for the locus. Conclusion:A novel type 2 tri-allelic pattern (8, 9, 10) at the D13S317 locus has been identified among the ethnic Han Chinese population. The pattern has not been transmitted to the female offspring, and has been included in the international STRBase database for the first time.

[Objective]To investigate the genetic polymorphism of 42 short tandem repeats(STRs),including 41 non-CODIS loci from the Shenzhen Han population and evaluate their potential values in forensic application.[Methods]In our research,the AGCU 21+1 STR kit and Microreader? 23sp Direct ID System were applied to analyze the polymorphism of STR loci from 435 unrelated individuals of Shenzhen Han population.Modified-Powerstates and Arlequin v3.5 software were used to analyze the allele frequencies and forensic parameters,and perform the Hardy-Weinberg equilibrium test.[Results]A total of 418 alleles were detected from 435 unrelated individuals in Shenzhen,all consistent with Hardy-Weinberg equilibrium(P>0.05/42),with the allele frequency ranging from 0.001 1 to 0.552 9.Besides,the discrimination power(DP)ranged from 0.798 8(D1S1627)to 0.968 6(D7S3048),the polymorphic information content(PIC)ranged from 0.568 0(D1S1627)to 0.859 8(D7S3048),and the heterozygosity(H)ranged from 0.627 6(D1S1627)to 0.878 2(D20S470).Among all the STRs tested in the study,both D1S1656 and D21S1270 have 16 alleles and show the highest polymorphism.In comparison,only five alleles were observed in the D4S2408 locus,which displays the least polymorphism.[Conclusions]The 42 autosomal STR loci with high genetic polymorphism in Shenzhen Han population showed potential as an effective means for individual identification and paternity testing,especially in the cases with single parent or mutation detected.The obtained information can provide basic data for STR population genetics.

Objective:To compare the clinical efficacy of rotator cuff repair combined with transposition of the long head of the biceps tendon in the treatment of massive rotator cuff tears with or without subscapularis injury.Methods:A total of 73 patients with massive rotator cuff tears who underwent arthroscopic rotator cuff repair combined with transposition of the long head of the biceps tendon in the Department of Upper Arm, Sichuan Orthopaedic Hospital from January 2019 to September 2022 were retrospectively analyzed, including 35 males and 38 females, aged 58.8±7.6 years (range, 46-73 years). There were 11 cases on the left side and 62 cases on the right side. Causes of injuries: 6 cases of car accidents, 13 cases of fall injuries, 54 cases of no obvious cause of injury. The patients were divided into subscapularis injury group (37 cases) and no subscapularis injury group (36 cases) according to the subscapularis injury or not. The visual analogue scale (VAS), Constant - Murley score, University of California Los Angeles (UCLA) shoulder score and range of motion were used to evaluate shoulder pain and functional improvement. MRI of the shoulder was taken at the final follow-up and the rotator cuff was assessed for retear using Sugaya grading.Results:All patients successfully completed the operation. The VAS score at 1 year postoperatively in the group without subscapularis injury was 0.58±0.87, which was less than 1.16±1.14 in the group with subscapularis injury ( t=-2.426, P=0.018). The Constant-Murley score and UCLA score at 1 year postoperatively in the group without subscapularis injury were 94.03±6.19 and 32.94±2.59, respectively, which were greater than those of the group with subscapularis injury, which were 86.51±11.96 and 30.38±3.33, and the difference was statistically significant ( P<0.05). Anterior flexion and body external rotation at 1 year postoperatively in the group without subscapularis injury were 137.22°±26.79° and 54.72°±13.20°, which were greater than those in the group with subscapularis injury of 121.08°±22.83° and 38.65°±17.19°, and the difference was statistically significant ( P<0.05), and the difference between the two groups in body internal rotation was not statistically significant ( P>0.05). The subacromial spacing at 1 year postoperatively was 9.4±1.3 mm in the group without subscapularis injury and 9.0±1.5 mm in the group with subscapularis injury, and the difference was not statistically significant ( t=1.318, P=0.192). There were 2 cases of rotator cuff retear 1 year after surgery without subscapular muscle injury and 5 cases of subscapular muscle injury. Conclusion:The clinical efficacy of arthroscopic rotator cuff repair combined with transposition of the tendon of the long head of the biceps tendon in the treatment of massive rotator cuff tears with or without subscapularis muscle injury is satisfactory, and the improvement of pain and function is more obvious in patients without subscapularis muscle injury.

Objective To explore whether hepatocyte Perilipin-2(Plin2)is involved in the development of fatty liver related to comparative gene identification-58(CGI-58)deficiency mice and compare the effects of Plin2 and Plin3 on lipid droplet formation and lipid accumulation.Methods Based on CGI-58Flox/Flox mice as animal model,the adeno-associated viruses targeting mouse liver,CGI-58 knockout and Plini2 knockdown were achieved by co-expression Cre protein and micro-RNA targeting Plin2(Mi-KD).Then CGI-58 deficiency mice were used as control(NC)to detect the differences in metabolic phenotype and liver pathology.AML-12 mouse hepatocytes were used as cellular model and interfered with siRNA to achieve Plin2/Plin3 knockdown in AML-12 cells.Lipid droplet formation and lipid accumulation were compared with Bodipy staining and enzyme colorimetry in basal condition or lipid-overloaded condition(OA inducement)after Plin2/Plin3 knockdown.Results Plin2 knockdown(Mi-KD)reduced PLIN2 protein level by＞99％in mouse livers.Mi-KD decreased hepatomegaly(P=0.019 5)and liver injury(P=0.000 4),while reduced the histological NAS score(P=0.000 2)and hepatic triglyceride content(P=0.016 6)in the CGI-58 deficiency female mice.Mi-KD prevented microvesicular hepatic steatosis in the CGI-58 deficient female mice.Plin3 knockdown significantly reduced the triglyceride content in basal condition of hepatocytes(P=0.001 4),and Plin2 knockdown just showed a decreased trend.Plin2 or Plin3 knockdown significantly reduced the triglyceride content separately in lipid-overloaded hepatocytes(P＜0.05).Conclusion Hepatocyte Plin2 is essential in the development of microvesicular hepatic steatosis caused by CGI-58 deficiency.Both Plin2 and Plin3 are involved in lipid droplet formation and lipid accumulation in hepatocytes,and Plin3 shows a stronger effect.

Pulmonary lymphangiomyomatosis(LAM)is a rare chronic progressive diffuse cystic lung disease that mainly occurs in women of reproductive age.Pulmonary hypertension is a rare complication of LAM.Currently,there is insufficient evidence on the epidemiology,pathogenesis and treatment strategy of LAM related pulmonary hypertension.We reported a case of a woman at reproductive age with shortness of breath and diagnosed with LAM by the combination of specific lung imaging features and serum vascular endothelial growth factor D.Precapillary pulmonary hypertension was confirmed by right cardiac catheterization.Her condition was stable with Sirolimus and home oxygen therapy.

Pulmonary hypertension is a rare complication of pneumoconiosis,which is caused by long term denture dusk contacting and poor protection.Here we reported a case,who was a denture technician and had been engaged in denture grinding for more than 10 years.According to the specific lung imaging findings and dust exposure history,she was diagnosed with interstitial lung disease and pneumoconiosis (probable).Precapillary pulmonary hypertension was confirmed by right cardiac catheterization.