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[Abastract] Objective To discuss the effects of sample storage time and temperature on lymphocyte subsets detected by flow cytometry.Methods Use flow cytometry to detect lymphocyte subsets of a total of 53 blood samples from hospitalized and out-patient patients in Qinghai Provincial People's Hospital from October 26, 2018 to March 30, 2019. The test was completed within 4 hours after sample collection, which is the control group. The treatment groups are as follows: pretreatment was completed within 4 hours and detected after saving samples at room temperature (group A) for 24 and 36 hours;the tests were performed after keeping samples at room temperature (group B) for 24, 48, 72 hours; completed detection after preserving samples in 4℃condition (group C) for 24, 48, 72 hours. Results In treatment group A, there was no significant difference in the results of lymphocyte subsets detected after 24 hours of storage compared with the control group(P>0.05);after 36 h of storage, CD3+and CD3+CD8+T lymphocytes percentages were significantly increased [(74.28 ± 11.31)％vs (73.78 ± 11.33)％, (32.15 ± 14.82)％vs (31.00 ± 14.79)％;all P<0.05], while CD19+cells percentage were markedly decreased [(15.60 ± 12.23)％vs (16.11 ± 12.38)％;P<0.05]. In group B, compared with the control group, there was no obvious change in lymphocyte subsets tested after 24 hours(P>0.05); after 48 hours of storage, CD19+cells percentage were observably reduced [(15.60 ± 12.09)％ vs (16.11 ± 12.38)％;P<0.05], while other results showed no obvious change (P>0.05); after 72 hours of storage, CD3+and CD3+CD8+T cells percentages elevated significantly [(75.78 ± 11.18)％vs (73.78 ± 11.33)％, (32.57 ± 14.90)％vs (31.00 ± 14.79)％;all P<0.05], and CD19+cells percentage decreased markedly [(14.89±11.92)％vs (16.11±12.38)％;P<0.05]. In group C, there also was no significant change in the results between detecting after 24 hours and the control group(P>0.05); after 48 hours of storage, CD3+CD8+T cells percentage elevated obviously [(32.03±14.95)％vs (31.00±14.79)％;P<0.05] and CD19+cells percentage decreased markedly [(15.32±11.97)％vs (16.11±12.38)％;P<0.05];after 72 hours of storage, CD3+and CD3+CD8+T cells percentages were significantly increased [(75.63±11.08)％vs (73.78± 11.33)％, (32.62 ± 14.98)％vs (31.00 ± 14.79)％;all P<0.05], while CD56+and CD19+cells percentages were reduced observably [(8.21 ± 6.52)％ vs (9.02 ± 6.80)％, (14.83 ± 11.79)％ vs (16.11 ± 12.38)％;all P<0.05]. Conclusions The blood samples can be kept at room temperature and the testshould be completed within 48 h if the detection of lymphocyte subsets by flow cytometry cannot be performed in time;for the samples that have been pretreated, detection can be completed after saving at room temperature for 24 h, and the above treatments had no significant effect on the inspection results.
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Objective@#To discuss the effects of sample storage time and temperature on lymphocyte subsets detected by flow cytometry.@*Methods@#Use flow cytometry to detect lymphocyte subsets of a total of 53 blood samples from hospitalized and out-patient patients in Qinghai Provincial People′s Hospital from October 26, 2018 to March 30, 2019. The test was completed within 4 hours after sample collection, which is the control group. The treatment groups are as follows: pretreatment was completed within 4 hours and detected after saving samples at room temperature (group A) for 24 and 36 hours; the tests were performed after keeping samples at room temperature (group B) for 24, 48, 72 hours; completed detection after preserving samples in 4 ℃ condition (group C) for 24, 48, 72 hours.@*Results@#In treatment group A, there was no significant difference in the results of lymphocyte subsets detected after 24 hours of storage compared with the control group(P>0.05); after 36 h of storage, CD3+and CD3+CD8+T lymphocytes percentages were significantly increased [(74.28±11.31)% vs (73.78±11.33)%, (32.15±14.82)% vs (31.00±14.79)%; all P<0.05], while CD19+cells percentage were markedly decreased [(15.60±12.23)% vs (16.11±12.38)%; P<0.05]. In group B, compared with the control group, there was no obvious change in lymphocyte subsets tested after 24 hours(P>0.05); after 48 hours of storage, CD19+cells percentage were observably reduced [(15.60±12.09)% vs (16.11±12.38)%; P<0.05], while other results showed no obvious change (P>0.05); after 72 hours of storage, CD3+and CD3+CD8+T cells percentages elevated significantly [(75.78±11.18)% vs (73.78±11.33)%, (32.57±14.90)% vs (31.00±14.79)%; all P<0.05], and CD19+cells percentage decreased markedly [(14.89±11.92)% vs (16.11±12.38)%; P<0.05]. In group C, there also was no significant change in the results between detecting after 24 hours and the control group(P>0.05); after 48 hours of storage, CD3+CD8+T cells percentage elevated obviously [(32.03±14.95)% vs (31.00±14.79)%; P<0.05] and CD19+cells percentage decreased markedly [(15.32±11.97)% vs (16.11±12.38)%; P<0.05]; after 72 hours of storage, CD3+and CD3+CD8+T cells percentages were significantly increased [(75.63±11.08)% vs (73.78±11.33)%, (32.62±14.98)% vs (31.00±14.79)%; all P<0.05], while CD56+and CD19+cells percentages were reduced observably [(8.21±6.52)% vs (9.02±6.80)%, (14.83±11.79)% vs (16.11±12.38)%; all P<0.05].@*Conclusions@#The blood samples can be kept at room temperature and the testshould be completed within 48 h if the detection of lymphocyte subsets by flow cytometry cannot be performed in time; for the samples that have been pretreated, detection can be completed after saving at room temperature for 24 h, and the above treatments had no significant effect on the inspection results.
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BACKGROUND: Studies have shown that umbilical cord mesenchymal stem cells are ideal seed cells for tissueengineering research.OBJECTIVE: To isolate, culture and identify tree shrew umbilical cord mesenchymal stem cells, in order toestablish a standardized tree shrew umbilical cord mesenchymal stem cell lines.METHODS: Caesarean-isolated tree shrew umbilical cord samples were used to isolate and culture umbilical cordmesenchymal stem cells using tissue explant adherent method. Flow cytometry assay was used to detect cellsurface markers. Osteogenic and adipogenic induction media were used to induce umbilical cord mesenchymalstem cells to differentiate into osteoblasts and adipocytes.RESULTS AND CONCLUSION: The cultured umbilical cord mesenchymal stem cells expressed CD90 and CD105 with the positive rate of 99.9% and 99.8% respectively. Hematopoietic stem cell marker CD34 expression ratewas 0.0% and the endothelial cell marker CD31 expression rate was 0.7%, in line with the characteristics of umbilicalcord mesenchymal stem cell surface markers. Calcium nodules by alizarin red staining and lipid droplets by oil red Ostaining were observed in the induced cells. These experimental findings indicated that umbilical cord mesenchymalstem cells from tree shrews capable of osteogenic and adipogenic differentiation were successfully isolated and cultured.
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Objective:To analyze the chicken egg-white extracts were co-cultured with cells whether elevated stem cells protein,whether the cells transformation into stem cell.Methods:Four kinds of cells,making a common culture,a 50% chicken egg-white extract co-cultured for 3 days,cells were collected and frozen at -80 degrees,sending the company to do stem cell protein microarray.Results:C57-BMSC has three proteins occurred statistically significant change , TS-UC-MSC has one proteins occurred a statistically significant change ,293T has one protein occurred a statistically significant change ,and 293T-GFP has one protein occurred a statistically significant change.Conclusion:50% chicken egg-white extract co-cultured cells,the cells occurred the phenomenon of transformation into stem cells.
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BACKGROUND:Systemic lupus erythematosus is an autoimmune disease characterized as an emergence of a variety of autoantibodies in serum and multi-system and multi-organ lesions. Currently, there is a lack of effective treatment options, and umbilical cord mesenchymal stem cel s are a promising therapy for systemic lupus erythematosus based on cel biological roles. OBJECTIVE:To observe the therapeutic efficacy of human umbilical cord mesenchymal stem cel transplantation in the treatment of systemic lupus erythematosus in mice. METHODS:Human umbilical cord mesenchymal stem cel s were isolated and cultured fol owed by labeling with DiR fluorescence. Experimental mice were divided into normal control group (C57BL mice), model control group (C57BL/lpr mice), low-, medium-and high-dose umbilical cord mesenchymal stem cel therapy groups (C57BL/lpr mice), with 10 mice in each group. Mice in the low-, medium-and high-dose groups were respectively injected 0.5×106, 1×106, 2×106 human umbilical cord mesenchymal stem cel s, once a week, for 3 consecutive weeks. At the end of treatment, blood samples were col ected to measure antinuclear antibody, anti-histone antibody, anti-double stranded DNA antibody changes;OPG and Foxp3 gene expression changes were detected by quantitative PCR method. RESULTS AND CONCLUSION:After treatment, the levels of anti-nuclear antibodies, anti-histone antibodies and anti-double stranded DNA antibodies in the peripheral blood of mice were al declined in the low-, medium-and high-dose groups, while the number of peripheral blood CD4+CD25+T cel s was significantly elevated. OPG and Foxp3 gene expression was also increased dramatical y in the low-, medium-and high-dose groups, which was similar to that in the normal control group and significantly different from that in the model control group (P<0.01). Experimental findings demonstrate that after transplantation of human umbilical cord mesenchymal stem cel s, al relevant indicators in C57BL/lpr mice recovered to the normal levels, and the high-dose treatment group had the most obvious effect.
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Objective To establish a method of identification of DKO mouse model of Duchenne muscular dystro-phy, and to assess the dystrophin regeneration after stem cell transplantation.Methods Heterozygous mice were mated and the resulting offspring were used to identify their genotype by SSP-PCR.The plasma creatine kinase level was measured by biochemical analyzer and histological changes in the DKO mice were analyzed using HE staining.Human umbilical cord mesenchymal stem cells were prepared and injected into the DKO mice hindlimb muscle, and dystrophin expression was de-tected by immunofluorescence staining at 2 months after injection.Results Mating of heterozygous mice generated three kinds of genotype offsprings, and 21.2%of the offsprings were identified as DKO genotype (285 bp) .DKO mice showed dystrophic symptoms, their plasma creatine kinase level was as high as 16988.52 ±617.48 IU/L, and significant histologi-cal changes including diverse myocyte sizes, numerous centrally nucleated cells and connective tissue proliferation or in-flammatory cells infiltration.Human dystrophin expression was detected in the DKO mouse hindlimb muscle at two months after injection of human umbilical cord mesenchymal stem cells.Conclusion DKO mouse genotype can be identified by SSP-PCR, and DKO mouse is an ideal animal model for studies of stem cell therapy for Duchenne muscular dystrophy.
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BACKGROUND: Rat models induced by unilateral nephrectomy combined with adriamycin are widely used in screening anti-drugs of glomerular sclerosis. However, few reports concerning this method on inducing rabbit model of glomerular sclerosisOBJECTIVE: To establish a rabbit model of glomerular sclerosis, and to observe the renal function and histopathological changes during model preparation. DESIGN, TIME AND SETTING: Randomized controlled animal experiment was performed in Laboratory Animal Center of Kunming General Hospital of Chengdu Military Region between December 2008 and May 2009. MATERIALS: Twenty-five Japanese big-ear rabbits, weighing 1.75-2.25 kg, half males and half females, were selected to establish glomerular sclerosis models, and randomly divided into normal (n=10) and model (n=15) groups. METHODS: The left kidney of rabbits in the model group was removed under anesthetized with 30g/L saline solution of sodium pentobarbital (1 mL/kg). Rabbits in the normal group underwent a similar surgical procedure without kidney removing. At 1 week after operation, 5 mg/kg adriamycin was injected into rabbits in the model group, 3 mg/kg adriamycin was reinjected 2 weeks later. Same volume of physiological saline was injected in the normal group. MAIN OUTCOME MEASURES: The renal blood biochemical indexes were detected prior to and at weeks 4, 6, and 8 after model preparation. One nephridial tissue was harvested from each group to undergo pathological observation at week 8 after the second medication. RESULTS: ①Kidney in the normal group presented slightly white, slightly tough texture with smooth surface. ②Pathological features of focal segmental glomerulosclerosis was showed in the model group under a light microscope, presented as extracellular matrix hyperplasia, mesangial region expansion, renal glomerular capillary wall ball mix adhesion, tubule degeneration, or even interstitial fibrosis, as well as interstitial infiltration of inflammatory cells. ③Compared to the normal group, the total protein and albumin was and decreased, triglyceride, total cholesterol, low density lipoprotein cholesterol, urea nitrogen, and creatinine were significantly increased in the model group. The urine protein content of rabbits in the model group was obviously increased at 4 weeks, gradually reached a platform after 8 weeks, which still greater than that of the normal group. ④SPECT showed that the glomerular filtration rate of the model group was notably decreased (33 mL/min) than the normal group (92.6 mL/min).CONCLUSION: The unilateral nephrectomy combined with adriamycin in rabbits results in the formation of glomerular sclerosis, renal function and 24 h urinary protein is more visible in the phase change.