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To establish a method for isolation, culture and identification of adipose-derived mesenchymal stem cells (ASCs) from the inbreed line miniature pig of Wuzhishan (ILMW). Methods: A total of 100 g adipose tissues were obtained from subcutaneous tissues of neck in six-month old healthy ILMW (3 samples, male). ASCs from ILMW (ILMW-ASCs) were isolated from adipose tissues through 0.1% collagenase digestion. The cells at the 3rd, 5th, 8th, 13th passages were collected. Cell morphology, size, phenotype, cell cycle, and apoptosis were monitored. Cell differentiation was induced and cell proliferation curve was drawn. Results: The ILMW-ASCs, fibroblast-like or whirlpool-like, began the adherence at 36 h and entered a logarithmic phase in the 5th day. Eighty percent of them were fused in the 7th day. The average diameter and volume of ILMW-ASCs were (17.00±0.54) µm and (2.58±0.24)×10-9 L, respectively. The expressions of CD29, CD44 and CD90 were positive, and there was no significant difference between the different passages (all P>0.05). The expressions of CD45, CD8a and HLA-DR were increased with the increase in passages after the 3th passage (all P<0.05). The adipogenic induction of ILMW-ASCs was observed by positive oil red O staining, and the osteogenic induction of ILMW-ASCs was determined by positive alizarin red staining. Apoptosis and senescence occurred in the 13 passage of ILMW-ASCs, and the proportion of S phase of cell cycle was lower than that in lower passages (all P<0.05). Conclusion: ILMW-ASCs are one of the best choice for porcine ASCs, which might provide a source of candidate stem cells for therapy of large animal disease models and tissue or organ repairment.
Subject(s)
Animals , Male , Adipose Tissue , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , Swine , Swine, MiniatureABSTRACT
OBJECTIVE@#To identify a rare large off ladder (OL) allele at the Penta E locus.@*METHODS@#Chelex-100 was used to extract DNAs from the blood samples. The PCR fragments were purified, extracted, and subjected to TA cloning and sequencing.@*RESULTS@#An OL allele was identified by the PowerPlex™ 21 at the Penta E locus, which was postulated as allele 26 based on assigned size. The OL allele was verified as a novel fragment containing 26 full AAAGA repeats.@*CONCLUSION@#OL alleles and microvariants should be verified by direct sequencing. Typing of OL alleles has significance for both daily work and forensic genetics.
Subject(s)
Alleles , Polymerase Chain Reaction , RubiaceaeABSTRACT
Objective To establish an ELISA detection method for human serum carbonic anhydrase(CA)Ⅱ antibody,and to evaluate the level of serum CA Ⅱ antibody in patients with hypertensive nephropathy, chronic glomerulonephritis,type 2 diabetic nephropathy and healthy people.Methods To establish the ELISA method using CA Ⅱ,anti-CA Ⅱ monoclonal antibody and enzyme labeled secondary antibody,the evaluation of the degree of precision and sensitivity,and stability and anti-interference performance of the ELISA were made.Results The detection accuracy of serum CA Ⅱ antibody ELISA was 6.0%,the precision between the batches was 8.6%,the sensitivity was 0.032,and with favorable anti-interference performance and stability. The level of serum anti-CA Ⅱ antibody was significantly higher in patients with chronic glomerulonephritis and 2-type diabetic nephropathy compared with the normal control(P<0.05).There was no significant differ-ence in serum CAⅡ antibody level between hypertensive nephropathy group and healthy control group(P>0.05).Conclusion It is feasible to establish a serum CAⅡ antibody ELISA method to meet the needs of clini-cal test.CAⅡ antibody may be used as autoantibody to participate in the development of chronic glomerulone-phritis and type 2 diabetic nephropathy.
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OBJECTIVE@#To establish a method to isolate, culture and identify bone marrow-derived mesenchymal stem cells (BM-MSCs) from inbreed line miniature pig of Wuzhishan (ILMW) in vitro, to compare the biological characteristics of BM-MSCs derived from different pigs, and to supply BM-MSCs for investigating the repair mechanisms of renal injury in ILMW aft er unilateral ureteral obstruction.@*METHODS@#Four or 10-months old ILMW (n=4 per group) were selected and 5 mL of bone marrow fluid was extracted at 1 cm position from iliac wing edge. Mononuclear cells were isolated by density gradient method, then were cultured in the complete medium containing 3 different kinds of fetal bovine serum (FBS) (10% FBS, 12% FBS or 15% FBS). The cells of Passage 1, Passage 3, Passage 5 or Passage 11 were collected to examine biological characteristics including morphology, phenotype, differentiation ability, growth curve and cell cycle.@*RESULTS@#The BM-MSCs were attached to the culture dishes, which were fibroblast-like or whirlpoollike. Primary cultured cells began the adherence at 18 h and entered a logarithmic phase in the 6th day. Eighty percent of them were fused in the 9th day. There were no obvious anomalies in the subcultured cells. The expressions in cell surface antigens of CD29, CD44 and CD90 were positive, while the expressions of CD34 and CD45 were negative. There was no statistically significant difference between cells from different generations (all P>0.05). Under condition of osteogenic induction, alizarin red staining was positive at the 18th day, and alcian blue staining was positive at the 21th day. Cell cycle examination showed that the rate of G0/G1 was about 81.45%.@*CONCLUSION@#BM-MSCs of ILMW has advantages of earlier adherent time, more active proliferation, shorter cell subculture cycle, and stable biological characteristics after subculture, which is one of the best kinds of BM-MSCs coming from swine in mainland of China.
Subject(s)
Animals , Cell Differentiation , Cell Division , Cell Separation , Cells, Cultured , China , Mesenchymal Stem Cells , Cell Biology , SwineABSTRACT
Objective To explore the pathogenic mechanism by detecting the expression of membrane glycoprotein in the platelets of nonmuscle myosin heavy chain 9 related disease (MYH9-RD)patients.Methods Periperal bloods were obtained from 11 MYH9-RD patients and 7 normal family members.Flow cytometry was used for detecting the expression of the platelet membrane glycoprotein including GP Ⅱ b/Ⅲa(CD41/61),GP Ⅰ a(CD49b),GP Ⅰ b/Ⅸ/Ⅴ (CD42a) GP Ⅰ b(CD42b) and GPⅣCD36).Results The expression fluorescence intensity of platelet membrane glycoprotein GP Ⅱ b/Ⅲ a CD41/61),GPⅠa(CD49b),GP Ⅰ b/Ⅸ/Ⅴ (CD42a) GP Ⅰ b(CD42b) and GPⅣ (CD36) are 653.7 ±192.7,420.0 ± 151.3,667.7 ± 371.3 and 236.4 ± 64.2 respectively,which are significantly higher than those in normal controls (406.7 ± 126.1,181.2 ± 29.3,271.4 ± 91.6 and 136.1 ± 23.5 ; P ＜ 0.01) ; The expression of GP Ⅰ a(CD49b) was lower in patients with MYH9-RD (139.1 ± 54.9) than that in normal controls (192.2 ± 143.4),but there was no significant difference (P ＞ 0.05).Conclusion In our study,the diverse clinical manifestations in patients with MYH9-RD is probably associated with the expression level of platelet membrane glycoprotein
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Objective To investigate the potential influence of TNF on the expression of protease activated receptor (PAR)-1,2,3 and 4 by using P815 mast cells. Methods After being challenged with various concentrations of TNF for 2 h, 6 h and 16 h, the P815 mast cells were treated with or without Triton X-100 and the PAR expressions were detected by flow cytometry and immunofluorescent microscopy. Results Compared with the corresponding controls, TNF concentration-dependently upregulated expressions of PAR-2 and PAR-4 both in Triton X-100-treated and the untreated groups, but had no significant effect on the expression of PAR-1 and PAR-3. Moreover, no significantly different expressions of TNF-induced PAR-1, 2, 3 were observed between Triton X-100-treated and the untreated groups, whereas Triton X-100-treated PAR-4 expressions were significantly enhanced by TNF compared with the Triton X-100-untreated ones. Conclusion TNF can up-regulate PAR-2, 4 expression of P815 mast cells but has little effect on the expression of PAR-1, 3 correspondingly. And Triton X-100 treatment had no significant effect on TNF-modulated expression of PARs in P815 mast cells.
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Objective To investigate the clinical features, effective treatment, survival and prognostic factors of second primary tongue squamous cell carcinoma (SPTSCC) after nasopharyngeal carcinoma (NPC) radiotherapy. Methods The clinical data of 35 cases with SPTSCC after NPC radiotherapy were analyzed retrospectively. Kaplan-Meier method, Log-Rank test and COX proportional hazard mode was performed for statistical analysis. Results 3-year and 5-year overall survival rates were 55 % and 47 %, respectively, lymph node metastasis rate was 5.71 %. Univariate analysis indicated that gender (χ2 = 8.89, P = 0.00), T classification (χ2= 5.58, P= 0.02), clinical stage (χ2 = 8.51, P= 0.04) and treatment methods (χ2 = 29.37, P = 0.00) were important factors of prognosis. Multivariate analysis showed that treatment methods (P = 0.00) and T classification (P = 0.03) were independent prognostic factors. Operative treatment group had better prognosis than the non-operative treatment group, the difference was statistically significant (P <0.05), male patients in the risk of SPTSCC was higher than the female patients, and the incidence of SPTSCC was increased along with extension of the time after NPC radiotherapy. Conclusion The rate of the lymph node metastasis is lower for SPTSCC after NPC radiotherapy and treatment patterns and T stage are independent prognostic factors. Long-term follow-up after NPC radiotherapy is necessary to the early diagnosis of SPTSCC, so that to give surgery or combined therapy with surgery in order to achieve a good effect.
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OBJECTIVE To investigate the use ofultrasonically activated scalpel (UAS) in open thyroid surgery. METHODS From February 2004 to March 2005, thyroidectomy were performed in 77 cases by the same operation team. Forty seven cases received UAS thyroidectomy and 30 cases underwent conventional thyroidectomy. The operation time, estimated blood loss in operation, postoperative draining volume within the 24 hours, the length of hospitalization and postoperative complications between two groups were compared. RESULTS The average operation time of lobectomy and total/subtotal thyroidectomy in the UAS group were 61?4.34 and 85?3.02 minutes, and in conventional group were 82?3.37 and 121? 2.51 minutes. There was a significant difference in average operation time (P0.05). CONCLUSION The use of UAS in thyroid surgery was safe and can reduce the operation time.