BACKGROUND:The cryopreservation technology enables tissues/cells to be stored for a long time in a low-temperature environment while maintaining the integrity of their activity and function,which is of great significance for the construction of cell therapy,tissue engineering and biological sample banks.Cryoprotective agents often contain dimethyl sulfoxide and serum.To avoid the toxic side effects of dimethyl sulfoxide,the complexity of serum components and immune responses,although some finished cryoprotective agents have been marketed,they are faced with many difficulties such as high cost and limited application.Therefore,there is an urgent need to develop a cryoprotective agent with clear components and the ability to solve the above problems.OBJECTIVE:To evaluate the effects of a novel cryoprotectant on cryopreservation efficiency of different tissue and cell sources.METHODS:By applying the novel cryoprotectant as an experimental group with the commercially available and widely used cryoprotectant(control group)to umbilical cord Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,NK and CIK cells,comparative analyses were conducted in terms of cell morphology,number,viability,surface markers,differentiation potential,and cell-killing toxicity assay before cryopreservation and after resuscitation thawing.We confirmed the cryopreservation effect of the new cryoprotectant and its potential application value.RESULTS AND CONCLUSION:(1)The novel cryoprotectant facilitated the normal growth of cryopreserved Wharton's jelly tissue upon recovery,exhibiting mesenchymal stem cell morphology.No significant differences were observed between the experimental and control groups in terms of cell recovery rate,surface markers,and differentiation potential.(2)There was no significant difference in the number and viability of cells between the experimental group and the control group after cryopreservation of cord blood/peripheral blood mononuclear cells,and the cryo-resuscitated cell numbers and viability of derived NK cells/CIK cells did not show significant difference between the experimental and control groups.(3)For NK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportion of CD56+CD16+cell subpopulations between the experimental group and the control group.For CIK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportions of CD3+CD8+and CD3+CD56+cell subpopulations between the experimental group and the control group.(4)In terms of cytotoxicity testing,when the effective-target ratio of immune cells and melanoma cell line Mel624 was 20:1,whether it was NK cells/CIK cells derived from cord blood or peripheral blood mononuclear cells,there was no significant difference in the tumoricidal activity of cells between the experimental group and the control group.These findings suggest that the novel cryoprotectant can replace existing commercially available and widely used cryoprotectants,and is applicable to Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,as well as NK and CIK cells,providing a solid technical foundation for the scaling,standardization,and commercialization of universal cryoprotectants.

BACKGROUND:The cryopreservation technology enables tissues/cells to be stored for a long time in a low-temperature environment while maintaining the integrity of their activity and function,which is of great significance for the construction of cell therapy,tissue engineering and biological sample banks.Cryoprotective agents often contain dimethyl sulfoxide and serum.To avoid the toxic side effects of dimethyl sulfoxide,the complexity of serum components and immune responses,although some finished cryoprotective agents have been marketed,they are faced with many difficulties such as high cost and limited application.Therefore,there is an urgent need to develop a cryoprotective agent with clear components and the ability to solve the above problems.OBJECTIVE:To evaluate the effects of a novel cryoprotectant on cryopreservation efficiency of different tissue and cell sources.METHODS:By applying the novel cryoprotectant as an experimental group with the commercially available and widely used cryoprotectant(control group)to umbilical cord Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,NK and CIK cells,comparative analyses were conducted in terms of cell morphology,number,viability,surface markers,differentiation potential,and cell-killing toxicity assay before cryopreservation and after resuscitation thawing.We confirmed the cryopreservation effect of the new cryoprotectant and its potential application value.RESULTS AND CONCLUSION:(1)The novel cryoprotectant facilitated the normal growth of cryopreserved Wharton's jelly tissue upon recovery,exhibiting mesenchymal stem cell morphology.No significant differences were observed between the experimental and control groups in terms of cell recovery rate,surface markers,and differentiation potential.(2)There was no significant difference in the number and viability of cells between the experimental group and the control group after cryopreservation of cord blood/peripheral blood mononuclear cells,and the cryo-resuscitated cell numbers and viability of derived NK cells/CIK cells did not show significant difference between the experimental and control groups.(3)For NK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportion of CD56+CD16+cell subpopulations between the experimental group and the control group.For CIK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportions of CD3+CD8+and CD3+CD56+cell subpopulations between the experimental group and the control group.(4)In terms of cytotoxicity testing,when the effective-target ratio of immune cells and melanoma cell line Mel624 was 20:1,whether it was NK cells/CIK cells derived from cord blood or peripheral blood mononuclear cells,there was no significant difference in the tumoricidal activity of cells between the experimental group and the control group.These findings suggest that the novel cryoprotectant can replace existing commercially available and widely used cryoprotectants,and is applicable to Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,as well as NK and CIK cells,providing a solid technical foundation for the scaling,standardization,and commercialization of universal cryoprotectants.

Objective:To explore the predictive value of ankle brachial index (ABI), HEART and thrombolysis in myocardial infarction (TIMI)scores for restenosis in patients with lower extremity arteriosclerosis obliterans (LEASO) after stent implantation.Methods:A retrospective study of 100 patients who were admitted to the Heyuan City People′s Hospital for stent implantation for LEASO from January 2015 to January 2020 and met the inclusion criteria were collected, and the patients were divided into restenosis group ( n=28) and patency group ( n=72) according to whether they were restenosis after operation. The clinical data of the two groups of patients were compared, including gender, age, disease course, history of cardiovascular and cerebrovascular diseases, smoking history, regular medication after surgery, number of stents, total length, bilateral lesions, total occlusion of lower extremities, length of vascular lesions, high-sensitivity C-reactive protein (hs-CRP), fasting blood glucose, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total cholesterol, fibrinogen, hemoglobin, platelet count and red blood cell count, and ABI, HEART, TIMI scores were compared, the relationship between ABI, HEART and TIMI scores and postoperative restenosis was further analyzed. Measurement data were expressed as mean±standard deviation ( ± s), and two independent samples t-test was used for comparison between groups; enumeration data were compared between groups by Chi-square test. Based on independent risk factors in multivariate Logistic regression analysis, a nomogram prediction model was constructed using R 3.3.2 software and software package rms. Correlations were analyzed using bivariate Pearson linear correlation analysis. The receiver operating characteristic curve (ROC) was used to evaluate the predictive value of each score and nomogram model for postoperative restenosis, and the results were expressed as the area under the curve (AUC) and the 95% confidence interval (95% CI) of the area. Results:Patients in the restenosis group and the patency group had a history of smoking, the proportion of irregular postoperative medications, the number of implanted stents, the total length of implanted stents, hs-CRP, fibrinogen, total cholesterol, red blood cell count levels, and ABI, HEART, TIMI scores were significantly different ( P<0.05). Pearson correlation analysis showed that ABI score ( r=-0.527, P=0.001) was negatively correlated with postoperative restenosis, and the HEART score ( r=0.604, P=0.010) and TIMI score ( r=0.719, P=0.005) were positively correlated with postoperative restenosis. A history of smoking, irregular medication after surgery, number of implanted stents>2, total length of implanted stent>212 mm, hs-CRP>2 mg/L, fibrinogen>3.7 g/L, ABI score ≤0.58, HEART score>6.30, and TIMI score>4.30 were independent risk factors for postoperative restenosis. The ABI, HEART, and TIMI scores predict the AUC of the ROC curve for postoperative restenosis of 0.813, 0.789, and 0.798, cutoff points were 0.58, 6.30 and 4.30 score, sensitivities were 95.64%, 91.17% and 92.51%, specificities were 64.18%, 55.43% and 58.62%, respectively. The consistency index (C-index) and ROC curve AUC of the nomogram prediction model were 0.811 (95% CI: 0.721-0.901) and 0.818 (95% CI: 0.732-0.904), respectively, indicating a good degree of discrimination. Conclusions:ABI, HEART and TIMI scores are independent risk factors for restenosis after stent implantation in LEASO patients. ABI, HEART and TIMI scores can effectively predict the risk of restenosis after stent implantation in patients.

Objective: To investigate the clinical effect and safety of long-acting pegylated interferon-α-2b (Peg-IFN-α-2b) (Y shape, 40 kD) injection (180 μg/week) in the treatment of HBeAg-positive chronic hepatitis B (CHB) patients, with standard-dose Peg-IFN-α-2a as positive control. Methods: This study was a multicenter, randomized, open-label, and positive-controlled phase III clinical trial. Eligible HBeAg-positive CHB patients were screened out and randomized to Peg-IFN-α-2b (Y shape, 40 kD) trial group and Peg-IFN-α-2a control group at a ratio of 2:1. The course of treatment was 48 weeks and the patients were followed up for 24 weeks after drug withdrawal. Plasma samples were collected at screening, baseline, and 12, 24, 36, 48, 60, and 72 weeks for centralized detection. COBAS® Ampliprep/COBAS® TaqMan® HBV Test was used to measure HBV DNA level by quantitative real-time PCR. Electrochemiluminescence immunoassay with Elecsys kit was used to measure HBV markers (HBsAg, anti-HBs, HBeAg, anti-HBe). Adverse events were recorded in detail. The primary outcome measure was HBeAg seroconversion rate after the 24-week follow-up, and non-inferiority was also tested. The difference in HBeAg seroconversion rate after treatment between the trial group and the control group and two-sided confidence interval (CI) were calculated, and non-inferiority was demonstrated if the lower limit of 95% CI was > -10%. The t-test, chi-square test, or rank sum test was used according to the types and features of data. Results: A total of 855 HBeAg-positive CHB patients were enrolled and 820 of them received treatment (538 in the trial group and 282 in the control group). The data of the full analysis set showed that HBeAg seroconversion rate at week 72 was 27.32% in the trial group and 22.70% in the control group with a rate difference of 4.63% (95% CI -1.54% to 10.80%, P = 0.1493). The data of the per-protocol set showed that HBeAg seroconversion rate at week 72 was 30.75% in the trial group and 27.14% in the control group with a rate difference of 3.61% (95% CI -3.87% to 11.09%, P = 0.3436). 95% CI met the non-inferiority criteria, and the trial group was non-inferior to the control group. The two groups had similar incidence rates of adverse events, serious adverse events, and common adverse events. Conclusion In Peg-IFN-α regimen for HBeAg-positive CHB patients, the new drug Peg-IFN-α-2b (Y shape, 40 kD) has comparable effect and safety to the control drug Peg-IFN-α-2a.

BACKGROUND:Adenovirus, expressing within a limited period, can limit the expression time and amount of target genes that is not conducive to ongoing experiments. Here, we select adenovirus as vectors for genetic recombination with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Trail) gene fragment.
OBJECTIVE:To explore the construction of recombinant lentiviral vector carrying Trail in rats
METHODS:The Trail gene was obtained:according to GenBank in rat Trail gene sequence (NM_145681.1), we designed the gene specific primers of Trail-Age I-F and Trail-AgeI-R, and used AgeI as enzyme cutting site. PCR was applied to amplify Trail gene from rat cDNA Library and construct recombinant plasmids after cutting Trail gene to be cloned into expression vector GV218 by AgeI. Recombinant plasmids were transfected into 293T cells by Lipofeetamine2000 encapsulated recombinant plasmid and auxiliary packaging carrier. The Trail protein of lentiviral plasmids was expressed. Fol owing virus col ection, we identified virus titer and extracted protein from cells to detect Trail expression by western blot assay.
RESULTS AND CONCLUSION:Screened positive Escherichia coli DH5a competent cells were sequenced with 861 bp, which was consistent with Trail nucleotide sequence in GenBank. After transfection 2 days, virus liquid was col ected and confirmed as recombinant plasmid including Trail gene by PCR and Trail proteins expressed in 293T cells by western blot assay. Hole dilution method and real-time fluorescent quantitative PCR determination showed that the virus titer was 2×109 TU/mL. In this study, recombinant lentiviral vector carrying Trail is successful y constructed by homologous recombination in Escherichia coli.