During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.
Humans ; Animals ; Gene Editing ; CRISPR-Cas Systems/genetics* ; HEK293 Cells ; Homologous Recombination ; DNA

We conducted a prospective study to assess the non-inferiority of adjuvant chemotherapy alone versus adjuvant concurrent chemoradiotherapy (CCRT) as an alternative strategy for patients with early-stage (FIGO 2009 stage IB-IIA) cervical cancer having risk factors after surgery. The condition was assessed in terms of prognosis, adverse effects, and quality of life. This randomized trial involved nine centers across China. Eligible patients were randomized to receive adjuvant chemotherapy or CCRT after surgery. The primary end-point was progression-free survival (PFS). From December 2012 to December 2014, 337 patients were subjected to randomization. Final analysis included 329 patients, including 165 in the adjuvant chemotherapy group and 164 in the adjuvant CCRT group. The median follow-up was 72.1 months. The three-year PFS rates were both 91.9%, and the five-year OS was 90.6% versus 90.0% in adjuvant chemotherapy and CCRT groups, respectively. No significant differences were observed in the PFS or OS between groups. The adjusted HR for PFS was 0.854 (95% confidence interval 0.415-1.757; P = 0.667) favoring adjuvant chemotherapy, excluding the predefined non-inferiority boundary of 1.9. The chemotherapy group showed a tendency toward good quality of life. In comparison with post-operative adjuvant CCRT, adjuvant chemotherapy treatment showed non-inferior efficacy in patients with early-stage cervical cancer having pathological risk factors. Adjuvant chemotherapy alone is a favorable alternative post-operative treatment.
Female ; Humans ; Uterine Cervical Neoplasms/drug therapy* ; Prospective Studies ; Quality of Life ; Neoplasm Staging ; Chemoradiotherapy ; Chemotherapy, Adjuvant/adverse effects* ; Adjuvants, Immunologic ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use* ; Retrospective Studies

Objective:To investigate the clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET) between donor and recipient. Methods:Pregnancy outcomes of 31 donors of infertility patients of ovarian hyperresponsiveness and 30 recipients receiving by IVF-ET from January 2011 to December 2016 in the Reproductive Center of Henan Provincial People's Hospital were retrospectively analyzed.Results:The implantation rate of frozen-thawed embryo transfer (FET) cycle (52.05% vs. 58.70%), the clinical pregnancy rate (69.23% vs.73.91%), the live birth rate per transplant cycle (61.54% vs.65.22%), the cumulative pregnancy rate per oocyte pick-up cycle (87.10% vs. 100.00%), the twin rate (40.74% vs. 48.39%), and the live birth rate (77.42% vs. 96.77%) were slightly higher in donor group than in recipient group, while there were no significant differences ( P>0.05). There were no significant differences in implantation rate (58.70% vs. 40.74%), clinical pregnancy rate (76.00% vs. 57.14%), early abortion rate (5.26% vs. 12.50%) and live birth rate per transplant cycle (72.00% vs. 42.86%) between group of <38 years old and group of ≥38 years old ( P>0.05). There were significant differences in cumulative pregnancy rate (100.00% vs. 66.67%) and live birth rate per oocyte pick-up cycle (94.74% vs. 50.00%) ( P=0.037, P=0.007). Conclusion:Oocyte donation IVF-ET is an effective method to assist infertility patients such as premature ovarian failure, perimenopausal period, chromosomal abnormalities, and repeated failure of IVF-ET. The success rate of pregnancy in the recipients of oocyte is similar to that in the donors. All different age groups of recipients can obtain good pregnancy outcomes, and the young recipients have a higher live birth rate.

Objective:To investigate the clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET) between donor and recipient. Methods:Pregnancy outcomes of 31 donors of infertility patients of ovarian hyperresponsiveness and 30 recipients receiving by IVF-ET from January 2011 to December 2016 in the Reproductive Center of Henan Provincial People's Hospital were retrospectively analyzed.Results:The implantation rate of frozen-thawed embryo transfer (FET) cycle (52.05% vs. 58.70%), the clinical pregnancy rate (69.23% vs.73.91%), the live birth rate per transplant cycle (61.54% vs.65.22%), the cumulative pregnancy rate per oocyte pick-up cycle (87.10% vs. 100.00%), the twin rate (40.74% vs. 48.39%), and the live birth rate (77.42% vs. 96.77%) were slightly higher in donor group than in recipient group, while there were no significant differences ( P>0.05). There were no significant differences in implantation rate (58.70% vs. 40.74%), clinical pregnancy rate (76.00% vs. 57.14%), early abortion rate (5.26% vs. 12.50%) and live birth rate per transplant cycle (72.00% vs. 42.86%) between group of <38 years old and group of ≥38 years old ( P>0.05). There were significant differences in cumulative pregnancy rate (100.00% vs. 66.67%) and live birth rate per oocyte pick-up cycle (94.74% vs. 50.00%) ( P=0.037, P=0.007). Conclusion:Oocyte donation IVF-ET is an effective method to assist infertility patients such as premature ovarian failure, perimenopausal period, chromosomal abnormalities, and repeated failure of IVF-ET. The success rate of pregnancy in the recipients of oocyte is similar to that in the donors. All different age groups of recipients can obtain good pregnancy outcomes, and the young recipients have a higher live birth rate.

OBJECTIVE: To analyze the clinical features and prognosis of cervical adenocarcinoma (AC) and adenosquamous carcinoma of cervix (ASC). METHODS: The clinical data of 237 patients, including 201 cases of AC and 36 cases of ASC (FIGO stage ⅠB1-ⅡA), who underwent surgery in Qilu Hospital between September 2007 and September 2016 were reviewed. Clinical features of two groups were compared, and Kaplan-Meier survival analysis was performed to evaluate the prognosis. RESULTS: A larger proportion of ASC patients had lymphovascular space invasion compared with AC patients (<0.01), but no significant differences were observed in the age, FIGO stage, size of tumor, depth of stromal invasion, parametrial invasion, lymphatic metastasis and risk grade between two groups (all >0.05). The 5-year overall survival rates of AC and ASC groups were 79.4% and 78.3%, and the 5-year recurrence-free survival rates were 77.4% and 73.0%. Among patients received concurrent chemoradiotherapy, the 5-year overall survival rates were 71.0% and 61.4%, and the 5-year recurrence-free survival rates were 68.8% abd 61.1%, respectively. No significant differences were observed in 5-year overall survival rates and recurrence-free survival rates between AC and ASC patients (all >0.05). CONCLUSIONS Lymphovascular space invasion was more likely to occur in patients with ASC, but there was no significant difference in the prognosis between AC and ASC patients.
Adenocarcinoma ; diagnosis ; mortality ; Carcinoma, Adenosquamous ; diagnosis ; mortality ; Female ; Humans ; Neoplasm Staging ; Prognosis ; Retrospective Studies ; Survival Rate ; Uterine Cervical Neoplasms ; diagnosis ; mortality

Objective To evaluate the application of pathological diagnosis by rapid paraffin sections in the diagnosis and treatment of cervical diseases. Methods A total of 176 cases from our hospital between September 2009 and January 2010 with abnormal cervical cancer screening (including abnormal cytology result and high-risk HPV continuous positive) were randomly divided into 2 groups. Eighty-seven cases of them whose biopsy were got by Belinson forceps under the direction of colposcopy with rapid paraffin sections by ultrasonic histopathological rapid processor and BT transparent agents were selected as group A, while 89 cases with conventional paraffin sections were selected as group B. The production time and quality for paraffin sections were analyzed in the two groups. Those diagnosed as cervical intraepithelial neoplasia (CIN)Ⅱ or even worse and some special patients with CIN Ⅰ in the two groups received surgery, including loop electrosurgical procedure (LEEP) ,cold knife conization (CKC),.hysterectomy or radical hysterectomy.Tissue obtained after surgery was sent for routine pathological examination. If the results of postoperative routine pathological examination were inconsistent with the rapid or routine biopsy pathological examination,the heavier results were regard as the final diagnoses. The pathological results and diagnose accordance rates were recorded and compared between group A and group B. Results The quality of sections in two groups were all satisfied or basically satisfied to meet the diagnostic requirements. There were statistically significant difference in average production time between group A and B (40 minutes vs 24 hours, P < 0. 05). Thirty patients in group A and 32 patients in group B received surgery. The coincidence rate of biopsy pathological results and final diagnoses were 93% (28/30) for group A and 91% (29/32) for group B, in which there were not statistically significant difference (P > 0. 05). Conclusion Bapid paraffin sections technology is safe, accurate and economical for rapid pathological diagnosis of cervical diseases, which is worthy for being widely used in hospitals.

Interleukin-21 (IL-21) is a recently discovered cytokine with immunomodulatory activity. It is a new member of the IL-2 family and mainly secreted by the activated CD4~+ T cells. IL-21 can promote proliferation, differentiation and function of T cells, natural killer (NK) cells, and B cells after binding with its receptor, thereby effectively strengthen the innate immunity and acquired immunity. The antitumor activity of IL-21 has been demonstrated in several tumor models. Its phase Ⅱ trials is being carried out in metastatic melanoma and renal cell carcinoma. IL-21 has a broad application prospects in the treatment of malignant tumors. Here, we review the anti-tumor effects of IL-21 and the application foreground of combination of IL-21 with other drugs in future tumor immunotherapy.

Objective To investigate whether the proteasomes inhibitor MG262 exerts its anticancer function by inducing apoptosis in human ovarian cancer cells,and whether the extracellular signal regulated kinase (ERK) signaling pathway is involved in the regulation of apoptosis induction.Method Human ovarian cancer cell line SKOV3 was incubated with different concentrations of MG262 for 24 and 48 hours.Cell viability was evaluated with 3-(4,5-dimethyhhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points of culturing.Flow cytometry was used to detect cell apoptosis rate.The expression of vascular endothelial growth factor (VEGF) was evaluated with western blot and enzyme-linked immtmosorbent assay (ELISA).Western blot was used to detect the expression of phosphorylated ERK(pERK) .Results The viability of SKOV3 cells was decreased by MG262 in a concentration-dependent fashion(P＜0.05).After 24 h incubation with MG262 at 1,10,20,40,60 and 80 nmol/L,the viability rates of SKOV3 were (94.6±3.1)%,(92.7±3.7)%,(89.5±7.7)%,(84.2±5.1)%,(82.0±7.4)%and(76.8±11.0) % respectively,and after 48 h incubation,those figures were further decreased to (91.3±10.1)%,(86.8±4.5)%,(74.6±4.2)%,(56.8±2.1)%,(49.3±4.5)% and (37.4±5.4) %,respectively(P＜0.05).Apoptosis rate of SKOV3 cells induced by MG262,PD98059 or their combination was (30.7±4.3)%,(26.8±8.6)% and (50.3±10.6)%,respectively,which were significantly different compared with controls (P＜0.05).In contrast to SKOV3 cells,apoptosis rate of 293T ceils induced by MG262,PD98059 or their combination was (14.5±5.3) %,(16.2±7.5) % and (10.8±7.3)%,respectively,which were not significantly different compared with controls (P＞0.05).pERK expression decreased gradually in a time-dependent manner. And wild-type p53 expression was not significantly different.There was no significant difference between experimental and control 293T cells(P＜0.05).In addition,MG262 down-regulated VEGF secretion and expression in SKOV3 ceils (P＜0.05).Conclusions Proteasome inhibitors can induce apoptosis and inhibit cell proliferation and angiogenesis through ERK signal pathway in SKOV3 cells.

Objective To investigate the proliferation and cytotoxicity of cytotoxic T lymphocyte (CTL) induced by ovarian cancer cells transfected with a CD 80 gene containing retroviral vector Methods The ovarian cancer cell line TYK cells were transfected with retro virus vector PLXSN hCD 80 After geneticin (G418) selection, the CD 80 expression of the transfectants was confirmed by flow cytometry CTL was induced by co culture of CD 80 expressing TYK cells (TYK hCD 80 ) and peripheral blood mononuclear cells (PBMC) in the presence of anti CD 3 monoclonal antibody (McAb) Proliferation of PBMC was measured using 3?H Thymidine incorporation assay The lysis activity of CTL toward tumor cells was determined using methyl thiazolyl tetrazolium (MTT) assay Results After transfection and G418 selection, the CD 80 expression was proved by flow cytometry The highest rate of CD 80 expression was 84 9% The TYK cell line expressing high CD 80 was named TYK hCD 80 In the presence of anti CD 3 McAb,TYK hCD 80 significantly enhanced proliferation of PBMC than TYK cells ( 3H Thymidine incorporation, (40 604? 842) vs (8 000? 594) cpm ( P