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Objective To investigate the effect of CD73 on the proliferation and function of rat cardiac myocytes (CMs) in vitro. Methods The primary rat CMs and H9C2 cells were cultured in vitro, and lentivirus CD73 overexpression vector and empty vector group were constructed and transfected into CMs and H9C2 respectively. Experimental groups: CD73 overexpression group (OE group) including CMs-OE group and CMs-NC group, and negative control group (NC group) including H9C2-OE grop and H9C2-NC group, 5 in each group. After transfection, the expression of CD73 gene was detected by Real-time PCR method, and the proliferation ability was detected by MTT method, and the proliferation curve, doubling time and pulsatility of CMs were detected by non-destructive cardiomyocyte function analyzer. Results After 72 hours of lentivirus transfection, CD73 mRNA level in the over expression group was significantly higher than that in the control group (P<0. 05); the proliferation ability of CMs and H9C2 at 3-4 days after transfection in OE group was larger than that in NC group (P<0. 05); the proliferation curve of CMs and H9C2 cells in OE group was higher than that in NC group, and the doubling time of CMs and H9C2 in OE group was lower than that in NC group 72 hours after CMs transfection, the beating rate of OE group was higher than that of NC group, the expression of T-box 5(TBX5) and the secretion of vasuular endothelial growth factor VEGF) in OE group were higher than those in NC group, while the secretion of hypoxia iducible factor-1α(HIF-1α) and tumor necrosis factor-α(TNF-α) was lower than that of NC group. Conclusion CD73 can promote the proliferation of CMs and H9C2, promote CMs pulsation, TBX5 expression and VEGF secretion, and inhibit the secretion of HIF-α and TNF-α.
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Objective To analyze the characteristics and variation of occipital protuberance and occipital bunning of Chinese Holocene population. Methods Based on 275 adult male skulls, Neolithic Age ( 49 cases ), Bronze and Iron Age (171 cases) and modern (55 cases), the occipital protuberance and occipital bunning were divided into several categories, and the occurrence rates were analyzed and compared among different time periods. Results The characteristics and variation of occipital protuberance and occipital bunning were also different in different ages. The degree of the occipital protuberance was more and more significant with the age, which changed greatly from Neolithic Age to Bronze and Iron Age. In the Holocene, most people did not appear occipital bunning on the skull, but the relatively significant occipital bunning appeared in the Bronze and Iron Age. Conclusion The variation of occipital protuberance may be related to the change of skull size and overuse of trapezius muscle in Holocene. The occipital bunning appearance may reflect the relative development speed of the brain and skull to some extent.
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Objective:To explore the efficacy of Liuwei Nengxiao capsule combined with fecal bacteria transplantation on chronic functional constipation with spleen Qi deficiency. Method:A total of 129 patients with chronic functional constipation of spleen Qi deficiency treated in Affiliated Hospital of Shandong University of Traditional Chinese Medicine were randomly divided into three groups by the computer numerical table method： fecal bacteria transplantation group， Liuwei Nengxiao capsule group， and Liuwei Nengxiao capsule combined with fecal bacteria transplantation group， with 43 cases in each group. Fecal bacteria transplantation group was treated with fecal bacteria， Liuwei Nengxiao capsule group was treated with Liuwei Nengxiao capsule， and Liuwei Nengxiao capsule combined with fecal bacteria transplanted group was treated with Liuwei Nengxiao capsule combined with fecal bacteria. The efficacy of the 3 groups was compared. Result:After treatment， the frequency and amplitude of intestinal electricity increased significantly in three parts， with statistically significant differences from before treatment （P<0.05）. After treatment， the frequency and amplitude of the transverse colon， sigmoid colon， and descending colon of patients in Liuwei Nengxiao capsule combined with faecal transplantation group were higher than those in faecal transplantation group and Liuwei Nengxiao capsule group （P<0.05）. After treatment， substance P （SP） and motilin （MTL） of Liuwei Nengxiao capsule combined with faecal transplantation group were higher than those of faecal transplantation group and Liuwei Nengxiao capsule group， while nitric oxide （NO） and vasoactive intestinal peptide （VIP） were lower than those of faecal transplantation group and Liuwei Nengxiao capsule group （P<0.05）. The number of bifidobacteria and lactobacillus after treatment was higher in three groups than before treatment （P<0.05）. The numbers of yeast and enterobacteria were lower than before treatment （P<0.05）. After treatment， the number of bifidobacteria and lactobacillus in Liuwei Nengxiao capsule combined with faecal transplantation group was higher than that in faecal transplantation group and Liuwei Nengxiao capsule group， whereas the numbers of yeast， and enterobacteria were all lower than those in bacterial transplantation group and Liuwei Nengxiao capsule group （P<0.05）. After treatment， the BSFS scores of three groups were higher than before the treatment， while Wexner continence grading scale （Wexner） constipation score and patient assessment of constipation quality of life questionnaire （PAC-QOL） score were lower than before treatment （P<0.05）. Bristol stool form scale （BSFS） score of Liuwei Nengxiao capsule combined with fecal bacteria transplantation group was higher than that of fecal bacteria transplantation group and Liuwei Nengxiao capsule group. Wexner constipation score and PAC-QOL score were lower than those of fecal bacteria transplantation group and Liuwei Nengxiao capsule group （P<0.05）. Conclusion:Fecal bacteria transplantation combined with Liuwei Nengxiao capsule can effectively promote the recovery of intestinal electrical function， improve the intestinal flora balance， reduce the intestinal oxidative stress response， and promote the disappearance of patients' symptoms in the treatment of elderly chronic functional constipation， and thus is worth further promotion in clinic application.
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Objective: To highlight the relationship between miR-503 and wound healing of diabetic foot ulcer (DFU). Methods: Microarray analysis was used to detect the dysregulated miRNAs between the DFU tissues and normal tissues. The expression of miR-503 in tissues and serum of patients with DFU was detected by qRT-PCR technique. Then, CCK-8 assay was applied to determine the cell proliferation. TUNEL assay was used for assessing the apoptosis of cells after treatment with miR-503. Possible correlation between miR-503 and fbillin1 (FBN1) was predicted according to data accessed on RNA22 website online, and was detected for confirmation by luciferase reporter assay. Results: Microarray analysis showed that miR- 503 was significantly decreased in the DFU tissues compared with normal tissues. While marked increase in the expression of miR-503 in tissues and serum of patients with DFU was confirmed by qRT-PCR technique. Then, CCK-8 assay indicated that transfection of miR- 503 mimic obviously accelerated the cell proliferation. However, TUNEL assays suggested that miR-503 mimic inhibited the apoptosis of cells to improve the survival of fibroblasts. Besides, miR-503 AMO played a role in fibroblasts of DFU tissues exactly countering to miR-503 mimic treatment. It was predicted that MiR-503 is a complementary to the FBN1 by RNA22. Besides, SiRNA-FBN1 promoted the proliferation, but brought down the apoptosis of fibroblasts. Conclusions: MiR-503 regulates the function of fibroblasts and wound healing of patients with DFU by targeting FBN1 directly which provids a novel and critical target for diagnosis and treatment of DFU.
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Objective:To highlight the relationship between miR-503 and wound healing of diabetic foot ulcer (DFU).Methods:Microarray analysis was used to detect the dysregulated miRNAs between the DFU tissues and normal tissues. The expression of miR-503 in tissues and serum of patients with DFU was detected by qRT-PCR technique. Then, CCK-8 assay was applied to determine the cell proliferation. TUNEL assay was used for assessing the apoptosis of cells after treatment with miR-503. Possible correlation between miR-503 and fbillin1 (FBN1) was predicted according to data accessed on RNA22 website online, and was detected for confirmation by luciferase reporter assay.Results:Microarray analysis showed that miR- 503 was significantly decreased in the DFU tissues compared with normal tissues. While marked increase in the expression of miR-503 in tissues and serum of patients with DFU was confirmed by qRT-PCR technique. Then, CCK-8 assay indicated that transfection of miR- 503 mimic obviously accelerated the cell proliferation. However, TUNEL assays suggested that miR-503 mimic inhibited the apoptosis of cells to improve the survival of fibroblasts. Besides, miR-503 AMO played a role in fibroblasts of DFU tissues exactly countering to miR-503 mimic treatment. It was predicted that MiR-503 is a complementary to the FBN1 by RNA22. Besides, SiRNA-FBN1 promoted the proliferation, but brought down the apoptosis of fibroblasts.Conclusions:MiR-503 regulates the function of fibroblasts and wound healing of patients with DFU by targeting FBN1 directly which provids a novel and critical target for diagnosis and treatment of DFU.
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Objective To investigate the expression of interleukin-17 (IL-17) in peripheral blood mononuclear cells (PBMC) of patients with multiple myeloma and analyse it's clinical significance.Methods Thirty patients with multiple myeloma in the Department of Hematopathy,the Third Affiliated Hospital of Xinxiang Medical University from January 2016 to January 2017 were selected as observation group;and thirty healthy donors whose gender and age machted with patients in the observation group were selected as control group.Peripheral blood mononuclear cells were obtained from subjects in observation group and control group by using the method of density gradient centrifugation.The content of IL-17 in supernatant of PBMC culture solution of subjects in the two groups was detected by enzyme linked immunosorbent assay and the expression of IL-17 mRNA in PBMC was detected by quantitative real-time polymerase chain reaction;the cellular morphology of PBMC of subjects in control group was observed by scanning electron microscope after co-culturing with the serum of patients in observation group.Results The content of IL-17 in supernatant of PBMC culture solution of subjects in the observation group and control group was (30.79 ± 4.96),(10.10 ± 4.15) ng · L-1 respectively;the content of IL-17 in supernatant of PBMC culture solution of subjects in the observation group was significantly higher than that in the control group (t =3.412,P ＜ 0.05).The expression of IL-17 mRNA in PBMC in the observation group and control group was 4.28 ± 1.34 and 2.45 ±0.95 respectively;the expression of IL-17 mRNA in PBMC in the observation was significantly higher than that in the control group (t =2.796,P ＜0.05).After co-culturing of PBMC of subjects in the control group with the serum of patients in the observation group,the morphology of PBMC changed obviously,and membrane desquamate,membrane disintegration,even membranolysis were observed in some cells with the prolongation of co-culture time.Conclusion The over-expression of IL-17 in PBMC of patients with multiple myeloma may play a certain role in the occurrence and development of this disease.
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<p><b>OBJECTIVE</b>To investigate the differentiation of human amniotic epithelial cells (hAECs) into insulin secreting cells (ISCs) in vitro.</p><p><b>METHODS</b>The hAECs were isolated from human amnion by trypsin digestion, and the phenotype of the isolated cells were identified by flow cytometry and immunocytochemical staining. The hAECs at passage 3 were treated with nicotinamide and N2 supplement to investigate their differentiation into ISCs. At different times after differentiation, the expression of insulin and beta2 microglobulin (beta2-MG) was determined by immunocytochemical staining, while the content of insulin in supernatant from cultured hAECs was detected by radioimmunoassay and the expressions of insulin, pancreatic and duodenal homeobox factor-1 (PDX-1) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>(1) hAECs expressed high percent of CD29, CD73, CD166 and CK19. (2) At 7, 14 and 21 days, the percentages of insulin-positive cells in induced groups were 74.00% +/- 1.73%, 75.33% +/- 1.15% (see symbol) 75.67% +/- 0.58% respectively, which were negative in control groups. (3) At 7, 14 and 21 days, contents of insulin in supernatant from induced groups were (328.47 +/- 3.22) microIU/ml, (332.26 +/- 1.22) microIU/ml and (329.68 +/- 2.57) microIU/ml respectively, they were significantly higher than those in control groups (All P < 0.01). (4) PDX-1 mRNA and beta2-MG were expressed before and after the induction of hAECs, but insulin mRNA was expressed only in the induced groups.</p><p><b>CONCLUSION</b>hAECs can differentiate into ISCs, having the potential application for therapy of type I diabetes.</p>
Subject(s)
Humans , Amnion , Cell Biology , Cell Culture Techniques , Cell Differentiation , Physiology , Cells, Cultured , Epithelial Cells , Cell Biology , Flow Cytometry , Homeodomain Proteins , Metabolism , Insulin , Metabolism , Insulin-Secreting Cells , Cell Biology , RNA, Messenger , Genetics , Trans-Activators , Metabolism , beta 2-Microglobulin , MetabolismABSTRACT
<p><b>BACKGROUND</b>As a new electroencephalogram (EEG) signal processing technique for monitoring the depth of anesthesia, entropy consists of two indices: reaction entropy (RE) and state entropy (SE). Our study compared entropy with classical bispectral index (BIS) in reduction of myoelectrical interference and noxious stimuli with EEG signals.</p><p><b>METHODS</b>Two hundred and eighty patients (ASA I-II, 18-60 years old) undergoing scheduled surgeries from seven medical centers were enrolled. Anesthesia induction was managed with propofol via the target-controlled infusion (TCI) system. The results of BIS, RE, SE, mean arterial pressure (MAP) and heart rate (HR) were recorded before anesthesia induction, at the moment of unconsciousness, before and 2 minutes after administration of muscle relaxant, and before and one and three minutes after the tracheal intubation.</p><p><b>RESULTS</b>The values of half maximum effective concentrations (EC50), 5% effective concentrations (EC05) and 95% effective concentrations (EC95) of propofol effect-site concentration at the onset of unconsciousness were 1.2 (1.1-1.3 µg/ml), 2.5 (2.4-2.5 µg/ml) and 3.7 (3.7-3.8 µg/ml), while those of the predicted plasma propofol concentration were 2.8 (2.7-2.9 µg/ml), 3.9 (3.8-3.9 µg/ml) and 4.9 (4.8-5.0 µg/ml), respectively. The values of BIS, SE and RE were 62, 59 and 63 when 50% of patients lost consciousness, and 79, 80, 85 and 42, 37, 44, respectively, when 5% and 95% of patients were unconscious. The values of BIS, RE and SE dropped two minutes after the injection of muscle relaxant, but there were no significant differences between RE and SE. MAP and HR increased visibly, which indicated a reaction to tracheal intubation; the values of BIS, RE and SE, however, did not display any significant changes.</p><p><b>CONCLUSIONS</b>This large-sample multicentric study confirmed the values of RE and SE as approximating BIS value, at the onset of unconsciousness during propofol TCI anesthesia. After elimination of myoelectrical activation, all values of RE, SE and BIS decreased significantly and the three indices were less sensitive to noxious stimuli than cardiovascular responses.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Anesthesia , Anesthetics, Intravenous , Pharmacology , Blood Pressure , Electroencephalography , Electromyography , Entropy , Heart Rate , Monitoring, Physiologic , Propofol , Blood , PharmacologyABSTRACT
<p><b>BACKGROUND</b>The percutaneous transcatheter closure of secundum atrial septal defect (ASD) is increasingly a widespread alternative to surgical closure. The aim of this study was to assess long-term results of percutaneous closure of secundum-type atrial septal defect (ASDII).</p><p><b>METHODS</b>Between January 2001 and December 2005, 61 patients underwent a successful percutaneous closure of ASDII; including 25 male and 36 female. All were included in the patient study and were followed up to monitor by electrocardiogram and echocardiography, at intervals of 3 days, 3 months, 6 months, 1 year, 2 years, and 5 years after operation.</p><p><b>RESULTS</b>Three days after percutaneous transcatheter septal closure (PTSC), the right atrium diameter, right ventricular end-diastolic left-right diameter and right ventricular end-diastolic volume (RVEDV) decreased significantly (P < 0.05). Right ventricular end-diastolic anteroposterior diameter (RVEDD), right ventricular end-systolic volume (RVESV) and right ventricular ejection fraction (RVEF) also decreased (P < 0.01). During the period from 3 to 6 months, the size of the right atrium and right ventricle returned to normal range. Three days after PTSC, the left ventricular end-diastolic diameter (LVEDD), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular-systolic volume (LVSV) and left ventricular ejection fraction (LVEF) were significantly increased (P < 0.05). At 1 year, the size of the left atrium, left ventricle and left cardiac function returned to normal range (P < 0.01). There were no deaths or significant complications during the study. At five year follow-up, all defects were completely closed and remained closed thereafter.</p><p><b>CONCLUSION</b>Transcatheter closure of ASDII effectively eliminated the abnormal shunt and, subsequently improved the dimensions of each chamber and cardiac function.</p>
Subject(s)
Adolescent , Adult , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Young Adult , Follow-Up Studies , Heart Septal Defects, Atrial , Diagnostic Imaging , General Surgery , UltrasonographyABSTRACT
<p><b>OBJECTIVE</b>To construct an expression vector of the gene encoding rat interferon-gamma-inducible protein (IP-10) and identify its expression in NIH 3T3 cells.</p><p><b>METHODS</b>IP-10 gene was amplified by polymerase chain reaction (PCR) and inserted into the eukaryotic expression vector pcDNA3.1(+). After identification by PCR, restriction endonuclease digestion and sequence analysis, the recombinant expression vector pcDNA3.1(+)-IP-10 was transfected into NIH 3T3 cells via liposome. Immunofluorescence method was used to confirm the expression of pcDNA3.1(+)-IP-10 in the transfected NIH 3T3 cells. The expression of IP-10 protein in the supernatant of the transfected cells was examined by Western blotting.</p><p><b>RESULTS</b>PCR, restriction endonuclease digestion and sequence analyses confirmed successful construction of the recombinant vector pcDNA3.1(+)-IP-10. Immunofluorescence assay identified the expression of pcDNA3.1(+)-IP-10 in NIH 3T3 cells, and the expression of IP-10 protein was detected by Western blotting in the supernatant of the transfected cells.</p><p><b>CONCLUSION</b>A eukaryotic expression vector pcDNA3.1(+)-IP-10 has been successfully constructed, which provides the basis for investigating the therapeutic effect of IP-10 on Th1 type autoimmune disease.</p>
Subject(s)
Animals , Mice , Rats , Chemokine CXCL10 , Genetics , Metabolism , DNA Restriction Enzymes , Metabolism , Fluorescent Antibody Technique , Gene Expression , Genetic Vectors , Genetics , NIH 3T3 Cells , Plasmids , Genetics , Transfection , MethodsABSTRACT
In order to fabricate the HLA-DQA1 genotyping chip and develop an integrated, parallel technical platform to type HLA system, a pair of primers and a set of probes were designed according to the sequences of HLA-DQA1 exon 2, where the polymorphism is concentrated. The oligonucleotide chip was made with the methods developed in our laboratory. The target DNA was asymmetrically amplified with the labeled sense primer. The signals were scanned and analyzed after the hybridization between microarray and PCR product. The allele types of the samples were identified. The result was verified by the standard DNA and DNA sequencing. The results showed that the genotyping was successfully carried out in 50 standard DNA samples and 50 clinical samples. Among them, results of the 50 standard DNA samples matched their templates. In the other 50 samples, results of the randomly selected 10 matched their sequencing results except that two of them got the incompletely result. In reproducible tests, the signal reappear rate was 95%. It is concluded that HLA-DQA1 genotyping by using our array system is simple and convenient with satisfied accuracy and reproducibility.
Subject(s)
Humans , Genotype , HLA-DQ Antigens , Genetics , Allergy and Immunology , HLA-DQ alpha-Chains , Oligonucleotide Array Sequence Analysis , Oligonucleotide Probes , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To develop a method to rapidly quantitate and detect deletion of mitochondrial DNA (mtDNA) by microarray technique as a tool to study its relationship to tumorigenesis.</p><p><b>METHODS</b>A modified PCR was used to amplify full length mtDNA sequence in two samples of normal human blood leukocytes and five samples of gastric cancerous tissues, which were simultaneously labeled with fluorescin. The amplified products were verified by polyacrylamide gel electrophoresis (PAGE) and silver staining. Then, 17 pairs of overlapping primers of mtDNA were designed and their PCR products were used as mitochondrial probes. They were spotted onto amino-slides as microarray and hybridized. Hybridization image was scanned with GeneTAC laser, mtDNA copy number was counted by ScanAnalyzer software.</p><p><b>RESULTS</b>PAGE analysis showed that the designed probes were quite reasonable and strongly specific. The modified PCR method was efficient to amplify the whole mitochondrial genome with high-yield specific bands. The hybridizing spots were distinct, and background was clear. The signals of negative probes were close to those of background, and there was no significant difference between them (P > 0.05). The results were identical to those in the designed experiment. There were no significant differences between the results when the same sample of blood leucocytes or cancer tissues repeatedly examined with the same positive probes (P > 0.05), while there were significant differences when different types of samples were examined (P < 0.01). The hybridizing signals were stable and most of the data distributed in the range of mean +/- 2xSD.</p><p><b>CONCLUSION</b>The method here reported can rapidly, correctly and massively determine whether there exist special deletion and/or quantitative changes of mtDNA in patients with tumors. It will be helpful for the study of the relationship between mtDNA alteration and tumor development.</p>
Subject(s)
Humans , DNA, Mitochondrial , Genetics , Electrophoresis, Polyacrylamide Gel , Gene Deletion , Oligonucleotide Array Sequence Analysis , MethodsABSTRACT
<p><b>OBJECTIVE</b>To study the relationship between hRad17 mRNA expression and clinicopathologic factors and lymph node metastasis of gastric cancer, and to assess the significance of predicting the extent of lymph node metastasis and prognosis.</p><p><b>METHODS</b>hRad17 mRNA expression was examined in matched primary lesions, normal gastric mucosa and lymph node metastatic lesions among 52 gastric cancer patients by reverse transcription polymerase chain reaction (RT-PCR), polyacrylamide gel electrophoresis (PAGE) and silver stain with the relation between hRad17 mRNA expression and clinicopathologic factors analyzed. At the same time, hRad17 mRNA expressions in 5 gastric benign lesions and SGC7901 gastric carcinoma cell lines were also examined.</p><p><b>RESULTS</b>The primary tumor samples (88.4% positive) showed a significantly higher level of hRad17 expression compared with matched normal tissue (76.9% positive) (P = 0.014), so did the lymph node metastatic samples (94.2% positive) (P = 0.001). The hRad17 mRNA expression showed a low level in benign lesions, but very high in SGC7901 cell line. The hRad17 mRNA expression showed a higher level in patients with the number of lymph node metastasis above 15 than below 15 (P = 0.02), so did the diffused growth than the mass-like growth (P = 0.04).</p><p><b>CONCLUSION</b>The method of PAGE and silver stain can improve the sensitivity of RT-PCR. The degree of lymph node metastasis and invasiveness of carcinoma cells are more serious in cases with hRad17 mRNA overexpression, and extensive lymph node dissection should be carried out for these patients. Examination of hRad17 expression by RT-PCR before surgery is indicated to arrive at an optimum treatment scheme and to estimate the prognosis.</p>