Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by ...Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.展开更多
Objective： To investigate the expression of aquaporin 1 in cervical squamous carcinomas （CSC） and cervical precancerous lesions, and the relationship between the tumor clinicopathological parameters, prognosis and ...Objective： To investigate the expression of aquaporin 1 in cervical squamous carcinomas （CSC） and cervical precancerous lesions, and the relationship between the tumor clinicopathological parameters, prognosis and the expression of AQP1. Methods： Immunohistochemical method （EliVision） was used to detect the expression of AQP1 in samples from 106 patients [20 with normal cervical tissue, 30 with cervical intraepithelial neoplasia （stage Ⅰ and Ⅱ） and 56 with CSC]. Survival analysis was performed by Kaplan-Meier method. Results： AQP1 protein was expressed in vascular endothelia of all samples. It showed upregulation of AQP1 expression in CSC. There was a significant difference between CSC and normal cervical tissues （P〈0.05）. AQP1 was expressed in some tumor cells and unexpressed in normal squamous epithelial cells. And APQl-expressing tumor cells were positively related to lymph node metastasis. Patients with APQl-expressing tumor cells had the lower survival rate than the ones without. Conclusion： Abnormal expression of AQP1 plays an important role in the development of CSC. Positive expression of AQP1 in tumor cells maybe enhances tumor metastasis and could be used as a marker for tumor prognosis.展开更多
Background： Thermothempy has already been proved effective for the treatment of various tumors, including glioma. This study was performed to determine whether tumor necrosis factor-alpha was involved in the regulati...Background： Thermothempy has already been proved effective for the treatment of various tumors, including glioma. This study was performed to determine whether tumor necrosis factor-alpha was involved in the regulation of this biological process. Methods： RT-PCR and immunocytochemistry were used to investigate the levels of tumor necrosis factor-alpha mRNA and heat shock factor-1 protein, respectively, in glioma cells. Radioimmunoassay was used to dynamically monitor contents of TNF-α in nutrient fluid for C6 cells after hyperthermia treatment. Crystal violet staining method was used to detect glioma invasiveness. Results： The most obvious increase of heat shock factor-1 protein and tumor necrosis factor -alpha mRNA in C6 cells were observed at 30 min and 60 min after hyperthermia, respectively. In addition, the radioactivity of tumor necrosis factor-alpha in C6 cells＇ culture fluid also reached peak at 120 min of hyperthermia. The glioma invasiveness decreases and the concentration of tumor necrosis factor-alpha reached the maximum at 120 min of hyperthermia. Conclusion： Our results showed that the hyperthermia-mediated glioma invasiveness decreases was due to accelerated release of tumor necrosis factor-alpha,which could cause the decreases of glioma invasiveness by promoting the release heat shock factor-1 from neurospongioma cells .展开更多
基金Supported by the National Science and Technology Major Project(2011ZX09102-010-04)
文摘Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.
基金the Fund of Science and Technology Development Program of Shaanxi Province (2005K09-G6, 2005K09-G9)
文摘Objective： To investigate the expression of aquaporin 1 in cervical squamous carcinomas （CSC） and cervical precancerous lesions, and the relationship between the tumor clinicopathological parameters, prognosis and the expression of AQP1. Methods： Immunohistochemical method （EliVision） was used to detect the expression of AQP1 in samples from 106 patients [20 with normal cervical tissue, 30 with cervical intraepithelial neoplasia （stage Ⅰ and Ⅱ） and 56 with CSC]. Survival analysis was performed by Kaplan-Meier method. Results： AQP1 protein was expressed in vascular endothelia of all samples. It showed upregulation of AQP1 expression in CSC. There was a significant difference between CSC and normal cervical tissues （P〈0.05）. AQP1 was expressed in some tumor cells and unexpressed in normal squamous epithelial cells. And APQl-expressing tumor cells were positively related to lymph node metastasis. Patients with APQl-expressing tumor cells had the lower survival rate than the ones without. Conclusion： Abnormal expression of AQP1 plays an important role in the development of CSC. Positive expression of AQP1 in tumor cells maybe enhances tumor metastasis and could be used as a marker for tumor prognosis.
基金Supported by the Scientific Research Foundation of Hebei Provincial Department of Healththe Project of Science and Technology Research andDevelopment Plan of Tangshan City,Hebei Province(NO.20110165,20120144)(10140201A-15)
文摘Background： Thermothempy has already been proved effective for the treatment of various tumors, including glioma. This study was performed to determine whether tumor necrosis factor-alpha was involved in the regulation of this biological process. Methods： RT-PCR and immunocytochemistry were used to investigate the levels of tumor necrosis factor-alpha mRNA and heat shock factor-1 protein, respectively, in glioma cells. Radioimmunoassay was used to dynamically monitor contents of TNF-α in nutrient fluid for C6 cells after hyperthermia treatment. Crystal violet staining method was used to detect glioma invasiveness. Results： The most obvious increase of heat shock factor-1 protein and tumor necrosis factor -alpha mRNA in C6 cells were observed at 30 min and 60 min after hyperthermia, respectively. In addition, the radioactivity of tumor necrosis factor-alpha in C6 cells＇ culture fluid also reached peak at 120 min of hyperthermia. The glioma invasiveness decreases and the concentration of tumor necrosis factor-alpha reached the maximum at 120 min of hyperthermia. Conclusion： Our results showed that the hyperthermia-mediated glioma invasiveness decreases was due to accelerated release of tumor necrosis factor-alpha,which could cause the decreases of glioma invasiveness by promoting the release heat shock factor-1 from neurospongioma cells .
