ObjectiveTo detect human papilloma virus (HPV)infection with fluorescent quantitative real-time polymerase chain reaction (FQ-PCR)in Minnan population, and explore the correlation between HPV infection and carcinogenesis of esophageal carcinoma (EC)of Minnan patients.
MethodsFQ-PCR was performed to examine HPV-6, HPV-11, HPV-16 and HPV-18 in 100 healthy Minnan people (healthy group, 66 males and 34 females with their age of 52.35±6.72 years)and 100 Minnan patients with squamous EC (EC group and tumor-adjacent normal tissue group, 64 males and 36 females with their age of 51.62±6.37 years)between October 2009 and December 2012.
ResultsThe incidences of HPV infection in 100 EC tissues, 100 tumor-adjacent normal tissues and 100 esophageal mucosa tissues of healthy people were 22/100, 8/100 and 6/100 respectively, which were statistically different (χ2=10.63, P < 0.01). Positive infection of HPV-6, HPV-11, HPV-16 and HPV-18 was observed in 11 cases, 11 cases, 14 cases and 15 cases in EC group respectively, 5 cases, 6 cases, 7 cases and 8 cases in tumor-adjacent normal tissue group respectively, and 5 cases, 5 cases, 6 cases and 6 cases in the healthy group respectively (P > 0.05). Positive HPV infection was observed in 1 patients with well differentiated squamous EC, 21 patients with moderately differentiated squamous EC and 5 patients with poorly differentiated squamous EC (P > 0.05).
ConclusionHPV infection may exist in tumor tissue of Minnan patients with squamous EC, and may be correlated with carcinogenesis and development of squamous EC.
Objective To investigate the biological response and chemotaxis of endothel ial cells on template materials with different protein concentrations on the same surface, to provide the evidence for deep understanding of chemical induced cell motil ity. Methods Microcontact printing technique was employed to fabricate template materials with four different concentrations of collagen (50, 100, 200, 300 μg/mL) on the same substrate. Scanning electron microscopy was employed to characterize the qual ity of polydimethylsiloxane (PDMS) stamp. Confocal laser scanning microscopy (CLSM) was util ized to characterize the absorption of different concentrations of FITC conjugated collagen (50, 100, 200, 300 μg/mL) on the substrates surfaces. Software was used to analyze the fluorescence intensity of adsorbed protein on the substrates. Albumin was then used to block the substrates for cell culture of human umbil ical vein endothel ial cells (hUVEC). Substrates with no collagen adsorption were used as control samples. The influence of different concentrations of collagen on the prol iferation of hUVEC was investigated via MTT assay at 6, 24, 48 and 72 hours of culture. The cytoskeletal structures of cells were characterized by CLSM. The cell’ s migration speed and absolute displacement were measured by path measurement of single cell after 24 hours of culture. Results Fabricated PDMS stamps with complete pattern were flat. Template substrates were fully covered with evenly distributed collagen protein. The fluorescence intensities were 38.51 ± 1.63, 55.21 ± 3.88, 73.17 ± 3.59, and 80.95 ± 1.12 in adsorbed FTIC conjugated collagen with 50, 100, 200 and 300 μg/mL, respectively. Endothel ial cells spread better on various substrates coated with collagen than those of control samples. The prol iferation of endothel ial cells on collagen coated substrateswas significantly higher than that of control group (P lt; 0.05). With collagen concentration increasing from 50 μg/mL to 300μg/mL, the prol iferation abil ities and absolute displacements of endothel ial cells significantly increased (P lt; 0.05). Except for the group with 300 μg/mL, the migration speed of endothel ial cells on collagen coated substrates was significantly lower (P lt; 0.05) than that of control group. However, the migration speed of endothel ial cells on collagen coated substrates significantly increased (P lt; 0.05) along with collagen concentration increasing from 50 μg/mL to 300 μg/mL. Conclusion It is feasible to acquire domains with different protein concentrations on the same substrate using microcontact printing technique for investigating cell’s chemotaxis.
【摘要】 目的 評價人乳頭狀瘤病毒(HPV)DNA檢測在宮頸癌篩查中的價值。 方法 采用第二代雜交捕獲(HCⅡ)技術和液基細胞學測試(LCT)2種方法,對1026例在婦科病中心就診的受檢者進行同步盲法檢測,同時進行陰道鏡檢查。以宮頸活檢組織病理學檢查結果為診斷標準。評價該方案在宮頸癌篩查中的應用價值。 結果 病理檢查結果顯示,宮頸上皮內瘤變(CIN)Ⅰ級152例,CINⅡ級108例,CINⅢ級109例,宮頸浸潤癌28例。篩查高危型HPV感染366例,陽性率3570%, 在不同宮頸病變中的陽性率分別是:宮頸癌9290%(26/28),CINⅢ900%(99/109),CINⅡ8890%(96/108),CINⅠ8750%(133/152)。高危HPV對宮頸高級別病變的敏感性、特異性、陽性預測值,陰性預測值分別是9860%、8610%、1480%和9980%;HPV與LCT聯合檢測(平行試驗)的以上各指標分別是10000%、8090%、1210%和10000%。 結論 高危型人乳頭狀瘤病毒檢測在宮頸癌前病變的篩查中有較高的敏感度和陰性預測值,聯合LCT檢測是目前宮頸癌篩查具有診斷價值的方法。【Abstract】 Objective To investigate the value of high risk human papillomavirus(HPV) DNA dectection for cervical cancer screening. Methods Hybrid capture Ⅱ(HCⅡ)human papillomavirus (HPV) test and liquid based cytology test (LCT) were performed in 1026 patients treaed in Xuzhou No.1 hospital from May 2008 to May 2009,and the abnomal cytological or HPV DNA findings were further biopsied under the colposcopeto to appraise the appicational importance of each approach for screening cervical cancer. Results Pathological results showed that cervical intraepithelial neoplasial(CIN)Ⅰin 152 patients,CIN Ⅱ in 108 patients,CIN Ⅲ 109 patients,invasive cervical cancer in 28 patients.HPV infected 366 patients in detection, with 3570% positive rate. The infection rate of HPV in cervical cancer was 929%(26/28),in CIN Ⅲ was 908%(99/109),in CIN Ⅱ was 889%(96/108),and in CIN Ⅰwas 875%(133/152).The pathological results treated as standard,the sensitivity, soecificiy, positive prevalue, negative prevalue of HCⅡ HPV for detecting highgrade cervical lesions were 986%,861%,148% and 998%.The values for HPVLCT parallel test were 1000%,809%,121% and 100%. Conclusion Highrisk HPV DNA test is of high sensitivity and negativepredictive value. The combination of HCⅡ HPV and LCT tests are of great value for screening cervical cancer at present.
Objective To study the time effect of the gene expression of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes so as to lay a theoretical foundation for gene therapy of osteonecrosis. Methods The best multipl icity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 × 106 cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sl iced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo. Results The best MOI of BMSCs transfected with rAAV was 5 × 104 v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the b level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and b expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 ± 75.58) pg/mL and (4.80 ± 0.61) ng/mL respectively in experimental group 1, and were (32.28 ± 8.42) pg/mL and (0.64 ± 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.
Objective To transplant intravenously human brain-derived neurotrophic factor (hBDNF) genemodified bone marrow mesenchymal stem cells (BMSCs) marked with enhanced green fluorescent protein (EGFP) to injured spinal cord of adult rats, then to observe the viabil ity of the cells and the expressions of the gene in spinal cord, as well as theinfluence of neurological morphological repairing and functional reconstruction. Methods Ninety-six male SD rats weighing (250 ± 20) g were randomly divided into 4 groups: hBDNF-EGFP-BMSCs transplantation group (group A, n=24), Ad5-EGFPBMSCs transplantation group (group B, n=24), control group (group C, n=24), and sham operation group (group D, n=24). In groups A, B, and C, the spinal cord injury models were prepared according to the modified Allen method at the level of T10 segment, and after 3 days, 1 mL hBDNF-EGFP-BMSCs suspension, 1 mL Ad5-EGFP-BMSCs suspension and 1 mL 0.1 mol/L phosphate buffered sal ine (PBS) were injected into tail vein, respectively; in group D, the spinal cord was exposed without injury and injection. At 24 hours after injury and 1, 3, 5 weeks after intravenous transplantation, the structure and neurological function of rats were evaluated by the Basso-Beattie-Bresnahan (BBB) score, cortical somatosensory evoked potential (CSEP) and transmission electron microscope. The viabil ity and distribution of BMSCs in the spinal cord were observed by fluorescent inverted phase contrast microscope and the level of hBDNF protein expression in the spinal cord was observed and analyzed with Western blot. Meanwhile, the expressions of neurofilament 200 (NF-200) and synaptophysin I was analyzed with immunohi stochemistry. Results After intravenous transplantation, the neurological function was significantly improved in group A. The BBB scores and CSEP in group A were significantly higher than those in groups B and C (P lt; 0.05) at 3 and 5 weeks. The green fluorescence expressions were observed at the site of injured spinal cord in groups A and B at 1, 3, and 5 weeks. The hBDNF proteinexpression was detected after 1, 3, and 5 weeks of intravenous transplantation in group A, while it could not be detected in groups B, C, and D by Western blot. The expressions of NF-200 and synaptophysin I were ber and ber with transplanting time in groups A, B, and C. The expressions of NF-200 and synaptophysin I were best at 5 weeks, and the expressions in group A were ber than those in groups B and C (P lt; 0.05). And the expressions of NF-200 in groups A, B, and C were significantly ber than those in group D (P lt; 0.05), whereas the expressions of synaptophysin I in groups A, B, and C were significantly weaker than those in group D (P lt; 0.05). Ultramicrostructure of spinal cords in group A was almost normal. Conclusion Transplanted hBDNF-EGFP-BMSCs can survive and assemble at the injured area of spinal cord, and express hBDNF. Intravenous implantation of hBDNF-EGFP-BMSCs could promote the restoration of injured spinal cord and improve neurological functions.
Objective To clarify the trends of expression levels of several up-regulated micro RNA (miRNA) in tissues of atrophic bone nonunion and mRNAs and proteins of their related target genes in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and to explore their biological functions. Methods The hBMSCs were isolated from bone marrow of il iac bone by gradient centrifugation, and cultured. Osteogenic culture medium was used for osteogenic differentiation of the 4th generation of hBMSCs. The changes of corresponding miRNAs, mRNA and protein expression levels of related target genes were observed at 0 hour, 12 hours, 1 day, 2 days, 4 days, 7 days, and 14 days, by quantitative real-time PCR and Western blot. Results In the process of hBMSCs osteogenic differentiation, the mRNA and protein expression levels of osteoblastic target genes [alkal ine phosphatase l iver/bone/kidney (ALPL), bone morphogeneticprotein 2 (BMP-2), and platelet-derived factor alpha polypeptide (PDGF-A)] at most time points increased significantly whencompared with the values at 0 hour except that of BMP-2 decreased at 12 hours and 1 day, with maximum changes at 1 to 7 days. The miRNA expression levels, mRNA and protein expression levels changed significantly at different time points, while the trends of hsa-miRNA-149 and hsa-miRNA-654-5p changes were negatively correlated with the trends of ALPL and BMP-2 mRNA and protein expression changes respectively (P lt; 0.05). There was no obviously negative correlation between the trends of hsa-miRNA-221 change and PDGF-A change (P gt; 0.05). Conclusion In the osteogenic differentiation process of hBMSCs, hsa-miRNA-149 and hsa-miRNA-654-5p are closely related with the mRNA and protein regulation of ALPL and BMP-2, respectively.
ObjectiveTo summarize the biological characteristics of human epidermal growth factor receptor 2 (HER-2/neu) gene, the expression and meaning of HER-2/neu gene in gastric cancer, and clinical application of targeted medicine of HER-2/neu gene in gastric cancer.
MethodsRelated literatures about HER-2/neu gene and gastric cancer were retrieved for a review.
ResultsHER-2/neu gene encoded human epidermal growth factor receptor, and it participated in the gene regulation of tumor cell proliferation, invasion, and metastasis through the downstream signal transduction pathway. Amplification of HER-2/neu gene or overexpression of HER-2 was closely bound up to the occurrence and development of gastric cancer, however, whether it could be used as independent prognostic factors of gastric cancer remained to be controversial. Several targeted medicine of HER-2/neu gene had applied to clinical at present, and all of them obtained good short-term effect.
ConclusionHER-2/neu gene is a reliable target of gastric cancer and targeted medicine of HER-2/neu gene has a promising prospect.
ObjectiveTo prepare polyurethane (PU) microspheres and evaluate its physicochemical properties and biocompatibility for biomedical applications in vitro.
MethodsThe PU microspheres were prepared by self-emulsification procedure at the emulsification rates of 1 000, 2 000, 3 000, and 4 000 r/min. The molecular structure was tested by Fourier transform infrared spectrometer and the surface and interior morphology of PU microspheres were observed by scanning electron microscopy (SEM). PU microspheres prepared at best emulsification rate were selected for the subsequent experiment. The human umbilical vein endothelial cells (HUVECs) were cultured and seeded on the materials, then cell morphology and adhesion status were observed by calcein-acetoxymethylester/pyridine iodide (Calcein-AM/PI) staining. The cells were cultured in the H-DMEM containing 10%FBS with additional 1% phenol (group A), in the extracts of PU prepared according to GB/T 16886.12 standard (group B), and in H-DMEM containing 10%FBS (group C), respectively. Cell counting kit 8 (CCK-8) assay was used to detect the cell viability. The blood compatibility experiments were used to evaluate the blood compatibility, the PU extracts as experimental group, stroke-physiological saline solution as negative control group, and distilled water as positive control group. The hemolytic rate was calculated.
ResultsThe SEM results of PU microspheres at the emulsification rate of 2 000 r/min showed better morphology and size. The microstructure of the PU was rough on the surface and porous inside. The Calcein-AM/PI staining showed that the HUVECs attached to the PU tightly and nearly all cells were stained by green. CCK-8 assays demonstrated that group B and group C presented a significantly higher cell proliferative activity than group A (P<0.05), indicating low cytotoxicity of the PU. The absorbance value was 0.864±0.002 in positive control group and was 0.015±0.001 in negative control group. The hemolysis rate of the PU extracts was 0.39%±0.07% (<5%), indicating no hemolysis.
ConclusionThe PU microspheres are successfully prepared by self-emulsification. The scaffold can obviously promote cell attachments and proliferation and shows low cytotoxicity and favorable blood compatibility, so it might be an ideal filler for soft tissue.
Objective
To study the effect of secreted frizzled-related protein 1 (SFRP1) on airway remodeling in chronic obstructive pulmonary disease (COPD).
Methods
Forty-five patients with stable COPD and 21 healthy controls were collected in Jiangsu Province Hospital from June 2014 to June 2016. The level of SFRP1 in serum was detected by ELISA. The expression of SFRP1, E-cadherin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (16HBEs) were detected by Western blot.
Results
The level of SFRP1 in serum of the COPD patients was significantly higher than that in the healthy controls [(80.65 ± 15.01) pg/ml vs. (23.89 ± 2.59) pg/ml, P<0.05]. There were negative correlations between serum SFRP1 levels and lung function parameters (FEV
1, FEV
1%pred, and FVC) in the COPD patients (correlation coefficient r value were –0.368 9, –0.382 6 and –0.417 3, respectively, all P<0.05). The level of serum SFRP1 in the COPD smokers was significantly higher than that in the COPD non-smokers [(97.74 ± 21.95) pg/mlvs. (52.51 ± 14.87) pg/ml, P<0.05]. The expression of SFRP1 could be up-regulated by cigarette smoke extract in 16HBEs. The expression of E-cadherin of 16HBEs was up-regulated by recombinant SFRP1, but the expression of α-SMA of 16HBEs was down-regulated by recombinant SFRP1.
Conclusion
SFRP1 may be involved in the pathogenesis of airway remodeling of COPD by regulating the expression of E-cadherin and α-SMA of bronchial epithelial cells.
ObjectiveTo investigate the effect of microRNA-135a (miR-135a) in human amnion mesenchymal stem cell exosome (hAMSC-Exo) on the migration of fibroblasts.MethodsThe hAMSC-Exo was extracted with exosomes separation kit and identified, the effect of hAMSC-Exo on fibroblasts migration was detected by scratch test. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the relative expression of miR-135a gene in hAMSC-Exo after overexpression of miR-135a. Scratch test was used to detect the effect of hAMSC-Exo on the migration of fibroblasts after overexpression and knockdown of miR-135a. Western blot was used to detect the migration related proteins of fibroblasts [large tumor suppressor 2 (LATS2), E-cadherin, N-cadherin, and α smooth muscle actin (α-SMA)] after overexpression and knockdown of miR-135a. The 293T cell exosomes and hAMSC-Exo were used as control.ResultshAMSC-Exos were extracted successfully. Scratch test results showed that hAMSC group had the strongest ability to promote fibroblasts migration, and GW4869 (exosome inhibitor) treatment group had reduced ability to promote fibroblasts migration. qRT-PCR test showed that the relative expression of miR-135a gene in hAMSC-Exo increased significantly after over expression of miR-135a. Scratch test results showed that after over expression of miR-135a, hAMSC-Exo enhanced the migration ability of fibroblasts, while after knockdown of miR-135a, hAMSC-Exo weakened the migration ability of fibroblasts. Western blot results showed that the expressions of E-cadherin, N-cadherin, LATS2 were down regulated and α-SMA was up regulated in each hAMSC-Exo treatment group when compared with 293T cell exosomes group; after over expression of miR-135a, hAMSC-Exo decreased the expressions of E-cadherin, N-cadherin, LATS2 and increased the expression of α-SMA; while after knockdown of miR-135a, the ability of hAMSC-Exo was weakened.ConclusionmiR-135a in hAMSC-Exo can promote fibroblasts’ migration, inhibit the expressions of E-cadherin, N-cadherin, LATS2, and promote the expression of α-SMA.
