Objective To explore the effect of 2-chloroethyl ethyl sulfide(CEES) poisoning on keratinocyte migration and the regulatory role of microRNA(miR)-34a.Methods MTS was used to detect the viability of cells exposed to CEES in order to select an appropriate dose of CEES exposure in this in vitro model.The protein level of keratin 5 and keratin 10 was detected to assess cell differentiation status .Scratch assay was applied to evaluate cell migration ,and miR-34a silencing in keratinocytes was achieved by transfecting chemically synthesized miR-34a specific miRNA inhibitor.t-ERK1/2 and p-ERK1/2 levels closely related to cell migration were detected using Western blotting .Results An in vitro CEES exposure model of keratinocytes was established at the optimal concentration of 0.5 mmol/L CEES in the viability test , and this dose was chosen to evaluate cell migration changes .The migration of cells was significantly inhibited 24 h after CEES exposure , accompanied by no changes in morphology and keratin 5/10 levels.Silencing of miR-34a significantly increased the migration of cells exposed to CEES , which could be blocked by adding 5 μmol/L U0126 , an ERK1/2 phosphorylation selective inhibitor.Conclusion Silencing of miR-34a can significantly increase keratinocyte migration and partially reverse the inhibition of CEES-caused migration , which could be mediated by ERK 1/2 pathway activation .

Objective To retrospectively analyse the detection results of human leukocyte antigen(HLA) B27,and discuss its diagnosis value in disease such as ankylosing spondylitis(AS).Methods HLA-B27 detection was performed on 1 335 suspected AS patients treated in the hospital from January 2015 to June 2016 by using multiparameter flow cytometry instrument.The test results showed 201 patients were diagnosed with AS(AS group),1 134 patients were diagnosed with rheumatoid arthritis and other related diseases (non AS group).The test results were shown by using mean fluorescence intensity and positive lymphocyte expression rate.n addition,120 healthy people were enrolled as the control group in the physical examination center of the hospital.Results The positive rate of HLA-B27 screening in suspected AS patients was 15.73% (210/1 335),and the positive rate of HLA-B27 screening in the control group was 2.50% (3/120).The positive rate of HLA-B27 screening,the rate of cell expression and the average fluorescence intensity of AS group were 92.03%(185/201),(85.34±17.99)%,8.74±4.20,significantly higher than the non AS group and the control group(P<0.01).There was no significant difference between the control group and the non AS group (P>0.05).HLA-B27 screening positive patients were mainly concentrated in adolescence,and the positive rate of male was higher than that of female (P<0.05).Conclusion Flow cytometry examination of HLA-B27 can provide important basis for the diagnosis and differential diagnosis of AS.

Objective To explore the effect of electroacupuncture on activation of microglia in peri-infarct cortex after cerebral isch-emia-reperfusion in rats. Methods 36 male Sprague-Dawley rats were randomly divided into sham group (n=12), model group (n=12) and electroacupuncture group (n=12). The latter two groups were occluded the left middle cerebral arteries with modified Longa's method for 2 hours and reperfused. The electroacupuncture group received electroacupuncture at Quchi (LI11) and Zusanli (ST36) acupoints for 3 days. The nerve cell damage in peri-infarct cortex was observed with HE staining, while the expression of ED1 was determined with immunohisto-chemical staining, and the expression of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and IL-6 were determined with Western blotting. Results The neurological deficits score improved significantly in the electroacupuncture group (P<0.05), with less nerve cell dam-age, less number of ED1 positive microglia (P<0.05) and less levels of TNF-α, IL-1βand IL-6 (P<0.05), compared with the model group. Conclusion The electroacupuncture at Quchi (LI11) and Zusanli (ST36) acupoints can protect brain from ischemia-reperfusion injury, which might be associated with inhibiting the microglial activation and proinflammatory response in peri-infarct cortex.

Objective To investigate the effects of electroacupuncture (EA) at Quchi (LI11) and Zusanli (ST36) acupoints on the ultrastructural structure of cortical neurons in peripheral area and the protein expression of caspase- 3, Bcl- 2, Bax in rats with cerebral ischemia- reperfusion injury. Methods 36 male adult Sprague-Dawley rats were randomly divided into sham operation group, model group, and electroacupuncture group, with 12 rats in each group. The model group and electroacupuncture group were performed with left middle cerebral artery occlusion (MCAO) according to the modified Longa' methods. The electroacupuncture group received electroacupuncture at Quchi (LI11) and Zusanli (ST36) on the paralyzed limb, for 30 minute. The neurobehavioral scores were recorded before and after treatment. The ultrastructural structure of cortical neurons was observed with transmission electron microscope (TEM). The protein expression of caspase- 3, Bcl-2 and Bax were detected by Western blotting technique. Results The neurobehavioral score was lower in the electroacupuncture group than in the control group (P<0.05). Compared with the model group, the chromatin of neurons was even relatively, and the number of mitochondria increased. The expression of Bcl-2 was higher and the expression of caspase-3 and Bax was lower in the electroacupuncture group than in the model group (P<0.05). Conclusion Electroacupuncture at Quchi (LI11) and Zusanli (ST36) acupoints can inhibit the neurons apoptosis in peripheral area through mitochondria-caspase-3 pathway.

OBJECTIVE: To explore the genetic basis of a patient with primary infertility due to loss of zona pellucida. METHODS: The proband and his parents were subjected to whole exome sequencing. Candidate variants were validated by Sanger sequencing and bioinformatics analysis. RESULTS: The proband was found to harbor compound heterozygous variants of the ZP1 gene in exon 5 c.874C>T(Gln292*) and exon 7 c.1127_1128delCT (p.Ala376GlyTer386), which were respectively inherited from her mother and father. CONCLUSION The compound heterozygous variant of ZP1 gene probably underlie the loss of zona pellucida in oocyte disease in the proband.

Objective To investigate expression and significance of decorin(DCN)in liver tissue and serum of liver transplant patients with chronic rejection(CR).Methods Immunohistochemistry(SP method)was used to detect expression of DCN in liver tissue of 16 normal controls, 20 patients with cirrhosis, 46 liver translantion patients without CR and 8 patients with CR.Enzyme-linked immunosorbent assay method(ELISA method)was used to determined the content of DCN in serum of all research subjects.Results The expression of DCN was negative in normal hepatic tissues and with/without CR, cirrhosis tissues showed strong expression of DCN.The positive expression rate and the average optical density value of DCN in liver transplant tissues with CR had significant difference comparing with Cirrhosis tissues(25% vs 55%, 0.1249 ±0.0039 vs 0.2357 ±0.0396, P <0.01,while no statistic siqnificance compared to normal liver tissues and those without CR.The level of DCN in serum was significantly higher in liver transplant patients with CR, with significant difference comparing with normal people, liver cirrhosis and transplant liver patients without CR(54.0833 ± 6.0325)μg/L vs(1.0232 ± 0.9105)μg/L,(12.6202 ± 1.5370)μg/L,(17.7102 ± 2.3562)μg/L, P < 0.01).The concentration of DCN in serum showed a positive correlation with the degree of CR.Conclusions DCN showed negative expression in liver tissue and increased significantly in serum of liver transplantation patients with CR.This suggests that DCN may be involved in occurrence and development of CR.At the same time the determination of DCN in serum maybe become an important indicator of the early diagnosis, development and prognosis of CR for liver transplant patients.

Objective:To investigate the accuracy and application value of color Doppler flow imaging (CDFI) in diagnosis of carotid artery stenosis.Methods:A retrospective analysis was performed on the clinical data of 172 patients with cerebral infarction or transient ischemic attack (TIA) underwent both CDFI and cerebral digital subtraction angiography (DSA) in our hospital from January 2013 to July 2019. The carotid arteries from these patients were classified into 6 categories: normal type, mild stenosis (stenosis degree<30%), moderate stenosis (stenosis degree: 30%-69%), severe stenosis (stenosis degree: 70%-94%), subtotal occlusion (stenosis degree: 95%-99%), and total occlusion (stenosis degree: 100%). The detection rates of carotid artery stenosis by CDFI and DSA were compared; and using DSA result as the gold standard, the accuracy of CDFI in diagnosing carotid artery stenosis was discussed. Kappa analysis was used to evaluate the consistency of the two detection methods.Results:A total of 344 carotid arteries were detected in the 172 patients. CDFI examinations showed that 179 were normal, 15 were with mild stenosis, 66 were with moderate stenosis, 61 were with severe stenosis, 13 were with subtotal occlusion, and 10 were with total occlusion. DSA showed that 160 were normal, 32 were with mild stenosis, 84 were with moderate stenosis, 45 were with severe stenosis, 14 were with subtotal occlusion, and 9 were with total occlusion. The vascular stenosis detection rate of DSA was 53.5% (184/344), and that of CDFI was 48.0% (165/344), without significant difference ( P>0.05). Using DSA result as the gold standard, the accuracy, sensitivity and specificity of CDFI in diagnosing carotid stenosis were 94.5%, 89.7% and 100% respectively. Consistency analysis showed that Kappa was 0.846, and the consistency of DSA and CDFI detection results was high. Conclusions:CDFI is a non-invasive and cost-effective method for diagnosing carotid artery stenosis, enjoying high consistency with DSA. CDFI can be used as the first choice for preoperative screening and postoperative follow-up of carotid artery stenosis.

The present study aims to analyze sperm concentration trends among young and healthy Chinese adults in Wuhan, Central China, from 2010 to 2015. Semen analysis data from 9357 participants were collected and analyzed using a general linear model and the Cochran-Armitage trend test. A significant decline was observed in sperm concentration (β [standard deviation]: -1.53 [0.16]; P < 0.001). In addition, a decline in sperm density was observed by stratifying student versus nonstudent sperm donors and by analyzing the year of birth or birth year cohort of the participants. Furthermore, the percentage of participants with sperm densities of over 40 × 10⁶ ml^-1 significantly decreased with year. Notably, a dramatic decline in sperm density was recorded over the first 5 years of study. This research reported a decline in sperm concentration among young adults in Wuhan, Central China, in 2010-2015.
Adult ; Aging ; China/epidemiology* ; Cohort Studies ; Healthy Volunteers ; Humans ; Male ; Retrospective Studies ; Semen Analysis ; Sperm Count ; Tissue Donors ; Young Adult

Objective: To determine whether the adenine base editor (ABE7.10) can be used to fix harmful mutations in the human G6PC3 gene. Methods: To investigate the safety of base-edited embryos, off-target analysis by deep sequencing was used to examine the feasibility and editing efficiency of various sgRNA expression vectors. The human HEK293T mutation models and human embryos were also used to test the feasibility and editing efficiency of correction. Results: ①The G6PC3(C295T) mutant cell model was successfully created. ②In the G6PC3(C295T) mutant cell model, three distinct Re-sgRNAs were created and corrected, with base correction efficiency ranging from 8.79% to 19.56% . ③ ABE7.10 could successfully fix mutant bases in the human pathogenic embryo test; however, base editing events had also happened in other locations. ④ With the exception of one noncoding site, which had a high safety rate, deep sequencing analysis revealed that the detection of 32 probable off-target sites was <0.5% . Conclusion: This study proposes a new base correction strategy based on human pathogenic embryos; however, it also produces a certain nontarget site editing, which needs to be further analyzed on the PAM site or editor window.
Humans ; Gene Editing ; CRISPR-Cas Systems ; Adenine ; HEK293 Cells ; Mutation ; Glucose-6-Phosphatase/metabolism*

Umbilical cord mesenchymal stem cells (UC-MSCs) have been widely used in regenerative medicine, but there is limited research on the stability of UC-MSCs formulation during production. This study aims to assess the stability of the cell stock solution and intermediate product throughout the production process, as well as the final product following reconstitution, in order to offer guidance for the manufacturing process and serve as a reference for formulation reconstitution methods. Three batches of cell formulation were produced and stored under low temperature (2-8 ℃) and room temperature (20-26 ℃) during cell stock solution and intermediate product stages. The storage time intervals for cell stock solution were 0, 2, 4, and 6 h, while for intermediate products, the intervals were 0, 1, 2, and 3 h. The evaluation items included visual inspection, viable cell concentration, cell viability, cell surface markers, lymphocyte proliferation inhibition rate, and sterility. Additionally, dilution and culture stability studies were performed after reconstitution of the cell product. The reconstitution diluents included 0.9% sodium chloride injection, 0.9% sodium chloride injection + 1% human serum albumin, and 0.9% sodium chloride injection + 2% human serum albumin, with dilution ratios of 10-fold and 40-fold. The storage time intervals after dilution were 0, 1, 2, 3, and 4 h. The reconstitution culture media included DMEM medium, DMEM + 2% platelet lysate, 0.9% sodium chloride injection, and 0.9% sodium chloride injection + 1% human serum albumin, and the culture duration was 24 h. The evaluation items were viable cell concentration and cell viability. The results showed that the cell stock solution remained stable for up to 6 h under both low temperature (2-8 ℃) and room temperature (20-26 ℃) conditions, while the intermediate product remained stable for up to 3 h under the same conditions. After formulation reconstitution, using sodium chloride injection diluted with 1% or 2% human serum albumin maintained a viability of over 80% within 4 h. It was observed that different dilution factors had an impact on cell viability. After formulation reconstitution, cultivation in medium with 2% platelet lysate resulted in a cell viability of over 80% after 24 h. In conclusion, the stability of cell stock solution within 6 h and intermediate product within 3 h meets the requirements. The addition of 1% or 2% human serum albumin in the reconstitution diluent can better protect the post-reconstitution cell viability.