Objective To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. MethodsThirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups (n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay.Results The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B (P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A (P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B (P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A (P<0.05). ConclusionUnder the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
摘要:目的： 檢測大腸癌組織中Kras基因的突變情況以指導臨床治療。 方法 ：通過提取15例大腸癌石蠟組織中的DNA并進行PCR擴增,之后采用國際金標準方法直接測序法進行檢測獲得突變信息。 結果 ：15例大腸癌石蠟組織樣本中Kras有4例發生突變,突變率為266%。值得注意的是發現一個新的突變位點密碼子42,并且與密碼子12突變共存。 結論 ：密碼子42的突變進一步證明Kras突變不僅局限于密碼子12,13,61,還有與密碼子12共存的42位突變。Abstract: Objective: To detect the mutation status of Kras gene in colorectal cancers and to assist the clinical treatments Methods : DNA was extracted from fifteen formalinfixed, paraffinembedded tumor samples of colorectal cancers, and then the fragments containing codons 12,13 and codon 61 were amplified by PCR The sequences were indentified by direct sequencing which is gold standard for the detection of mutation Results : In the 15 samples of colorectal cancer patients, 4 mutations were observed, with 2 in codon 12 and 2 in codon 13 Suprisingly, a novel point mutation at codon 42 of Kras was found, and coexisted with mutation in codon 12 Conclusion : Except for codons 12,13,61 mutation, Kras has other mutation at codon 42 with coexisted with codon 12 point mutation
Objective To construct lentiviral vector carrying the human hepatocyte growth factor (hHGF) gene, and then to get hHGF gene/modified bone marrow mesenchymal stem cells (BMSCs) by infecting the BMSCs. Methods The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with Age I digestion and gene recombinant, then was identified with PCR and sequencing. Mediated by Lipofectamine2000, the three plasmids system of lentiviral vector including pGC-E1-hHGF, pHelper 1.0, and pHelper 2.0 was co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by real-time quantitative PCR. The BMSCs were infected by the constructed lentivirus and the multipl icities of infection (MOI) was identified with fluorescent microscope, the efficiency of infection with flow cytometry (FCM) analysis, the hHGF level with ELISA analysis, and the expression of hHGF gene with RT-PCR. Results Lentiviral vector carrying hHGF gene was constructed successfully. The titer of lentivirus was 1 × 108 TU/mL. The infection efficiency of BMSCs by hHGF lentiviral was high and reached 98% by FCM, and the best MOI was 10. A great mount of green fluorescence was observed with the fluorescent microscope at 28 days after infection. Peak concentration of hHGF secreted by BMSCs/hHGF reached 40.5 ng/mL at 5 days. The concentration could maintain a high level until 28 days after infection. RT-PCR showed that BMSCs/hHGF could express hHGF gene. Conclusion By lentiviral vector, hHGF gene was integrated into BMSCs genome, and it can express stably.
Objective To construct the responsive plasmid PTRE-HIF-1αof Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the cDNA of HIF-1α, which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid PTRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing. The responsive plasmid PTRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid PTRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2Tet-on cells.
ObjectiveTo study the distributions of virulence genes of Klebsiella pneumoniae (KP) and the distribution of hypervirulent KP (HvKP), and assess the performance of a single gene to predict HvKP.MethodsPolymerase chain reaction (PCR) method was used to analyze 12 virulence-related genes (entB, irp2, iroN, iucA, mrkD, fimH, c-rmpA, p-rmpA2, p-rmpA, wzy-K1, allS and peg-344) and drug-resistance gene blaKPC among 376 clinical KP strains collected from January 2016 to December 2018. Sequence types (ST) of KP were determined after sequencing and comparison, following the detection of 7 house-keeping genes (gapA, infB, mdh, pgi, phoE, rpoB and tonB) by PCR method. Statistical analyses were made for the distributions of virulence genes of KP and the distribution of HvKP with GraphPad Prism 8 software.ResultsAmong the 376 KP strains, the positive rates of entB, irp2, iroN, iucA, mrkD, fimH, c-rmpA, p-rmpA2, p-rmpA, wzy-K1, allS and peg-344 were 100.0%, 76.9%, 22.1%, 28.2%, 97.6%, 97.1%, 1.6%, 24.5%, 21.0%, 7.4%, 4.8% and 31.6%, respectively. The positive rates of the aforementioned virulence genes in the blaKPC-positive group (n=167) were 100.0%, 94.0%, 7.2%, 16.8%, 97.0%, 96.4%, 0.0%, 15.0%, 6.6%, 0.0%, 0.0% and 21.0%, respectively, and those in the blaKPC-negative group (n=209) were 100.0%, 63.2%, 34.0%, 37.3%, 98.1%, 97.6%, 2.9%, 32.1%, 32.5%, 13.4%, 8.6% and 40.2%, respectively; there was no statistically significant difference in entB, mrkD or fimH between the two groups (P>0.05), the positive rate of irp2 was higher in the blaKPC-positive group than that in the blaKPC-negative group (P<0.05), and the positive rates of the rest virulence-related genes were lower in the blaKPC-positive group than those in the blaKPC-negative group (P<0.05). The rate of HvKP in the blaKPC-negative group was higher than that in the blaKPC-positive group (38.3% vs. 18.0%, P<0.05). As a marker of HvKP, iucA showed high sensitivity and specificity (90.9% and 97.7%), followed by p-rmpA2 (83.6% and 100.0%) and iroN (73.6% and 99.2%). ST11 accounted for 87.4% in the blaKPC-positive group, while ST23, ST20, ST54 and ST29 were the four primary types in the blaKPC-negative group, accounting for 23.4% totally.ConclusionsDifferent virulence genes mean different distributions in KP. blaKPC-negative KP is more virulent than blaKPC-positive KP. iucA and p-rmpA2 could serve as good predicators of HvKP. Armed with extreme virulence and drug-resistance, blaKPC-positive HvKP is of great clinical concern.
ObjectiveTo assess the association of vascular endothelial growth factor (VEGF) gene-460C/T and-634C/G polymorphism with diabetic retinopathy (DR) among patients in Asia and European by meta-analysis. MethodsA systematic search of electronic databases (PubMed, Cochrane Library, EMBASE, VIP, Wanfang technological, CNKI, etc.) was carried out until Jun, 2014. Case-control studies on the relationship between genetic polymorphism of VEGF-460C/T and VEGF-634C/G with diabetic retinopathy were included in this analysis. The data were quantitatively analyzed by RevMan 5.0 software after assessing the quality of included studies. The pooled odds ratios (OR) and their corresponding 95% confidence intervals (CI) were used to assess the strength of the association. ResultsVEGF-460C/T (7 studies:899 cases and 786 controls) and VEGF-634C/G (10 studies:1615 cases and 1861 controls) were inclued in this meta-analysis. Significant association was found for-460C/T polymorphism in Aisa (C versus T:OR=1.52, 95%CI was, Z=3.72, P=0.0002; CC versus CT+TT:OR=1.61, 95%CI was[1.22, 1.90], Z=3.05, P=0.002; TT versus CT+CC:OR=0.64, 95%CI was[1.19, 2.19], Z=2.07, P=0.04), and VEGF-634CC gene type was associated with DR in European (OR=1.56, 95%CI[1.08, 2.25], Z=2.37, P=0.02). No significant publication bias was found. ConclusionsThe meta-analysis demonstrated that DR was associated with VEGF-460C/T polymorphism in Asia, and C alleles and CC gene type was the risk polymorphism; VEGF-634C/G polymorphism was not associated with DR, but its CC genotype maybe the risk factor in European. Further case-control studies based on larger sample size are still needed, especially for-634C/G polymorphism.
ObjectiveTo explore the relationship between Beclin 1 level and lymph node metastasis in patients with non-small cell lung cancer.MethodA total of 204 surgical specimens of patients with non-small cell lung cancer from September 2011 to September 2016 were collected in our hospital. There were 116 males and 88 females . Beclin 1 levels were detected by Western blotting. There were 116 males and 88 females at average age of 55.3±11.2 years. The patients were divided into three groups including a group N0 (no lymph node metastasis), a group N1(intralobar and interlobar lymph node metastases, and no mediastinal lymph node metastasis), and a group N2 (mediastinal lymph node metastasis). The differences of Beclin 1 levels in tumor tissues and lymph nodes of patients with N0, N1 and N2 were statistically analyzed.ResultsAmong 204 patients of lung cancer, 36 patients were squamous cell carcinoma and 168 patients were adenocarcinoma. The levels of Beclin 1 in tumor tissues of N0, N1 and N2 groups decreased gradually with a statistical difference (P<0.05). In the three groups, the levels of Beclin 1 in the lung hilum and intrapulmonary lymph nodes (N1 Beclin 1) of N1 and N2 groups were less than that of N0 group with a statistical difference (P<0.01). In the three groups, the level of Beclin 1 in the mediastinal lymph nodes (N2 Beclin 1) of N2 group was less than that of the N0 and N1 groups with a statistical difference (P<0.01). In the N1 group, the level of N1 Beclin 1 was less than that of N2 group (P<0.01). In the N2 group, though the level of N1 Beclin 1 was less than N2 Beclin 1, there was no statistical difference (P>0.05). ConclusionBeclin 1 level can be used as a reference index to judge the benign and malignant lung masses, and lymph node Beclin 1 level can be used as an important reference index to help determine whether there is lymph node metastasis in lung cancer.