Objective To develop and optimize a new method to extract miRNAs from plasma.Methods miRNAs were extracted from plasma by mixing it with the extraction solution that contained surfactant and by heating .Then the ribonuclease inhibitor was added into the extraction to prevent RNAs from degradation .The expression level of each miRNA was detected by real-time quantitative PCR in oder to evaluate the feasibility of this method .Results A method which extracted miRNAs from plamsa in just one step was established .The specificity , reproducibility and stability of this method have been demonstrated by real-time quantitative PCR .Conclusion The one-step method is simple , inexpensive , and plasma-saving.It seems like a new method for clinical examination of miRNAs from plasma .

Objective To establish RNase L gene knockout HEK 293 cell lines using CRISPR/Cas9 system.Methods Small guide RNA ( sgRNA) sequences of human RNase L were designed and sgRNAs were inserted into pCas-Guide and pCas-guide RNA(gRNA) vectors were obtained.The donor DNA sequences of the homologous arm were designed for RNase L knockout .In the presence of the right homologous arm , the resistance gene of hygromycin B and the left homologous arm as templates of homology-directed repair , the donor DNA template was amplified by overlopping PCR and cloned into the pBackZero-T expression vector and pBackZero-T-RNase LK vector was obtained .The pCas-gRNA vector and pBackZero-T-RNase LK vector were co-transfected into HEK293 cells to establish the stable expression cell line of RNase L gene knockout .Cells were cultured with hygromycin B , while Western blotting and DNA sequencing were used to analyze the gene of RNase L knockout from genome .Results and Conclusion The pCas-gRNA vector and pBackZero-T-RNase LK vector were successfully constructed.Five RNase L gene knockout HEK293 cell lines were generated,contributing to the study of the biological function and molecular mechanism of RNase L .

Objective To observe the effect of different doses of atorvastatin combined with probucol on contrast induced acute kidney injury (CIAKI) and serum uric acid in elderly patients.Methods Totally 121 cases admitted for coronary angioplasty were randomly divided into three groups.In standard combining treatment group (n＝35),atorvastatin 20 mg qn and probucol 0.25 g,tid were given with no loading dose intake before angioplasty.In intensively combined treatment group (n＝41),atorvastatin 40mg qn and probucol 0.25 g,tid were given with a loading dose of atorvastatin 40 mg and probucol 0.5 g at 2 hours before angioplasty.In intensive atorvastatin therapy group(n＝45),atorvastatin 40 mg qn were given,with a loading dose of atorvastatin 40 mg 2 hours before angioplasty.All patients were then evaluated 24 hours before and after angioplasty procedure,and their blood urea nitrogen (BUN),serum creatinine (Scr),serum uric acid (SUA),estimated glomerular filtration rate (eGFR) by modified diet in renal disease study (MDRD) method were tested.The serum and urine at 24 hours before and after operation were collected.Neutrophil gelatinase associated lipocalin (NGAL) were determinated by enzyme linked immunosorbnent assay (ELISA) method.Results After operation,eGFR was decreased in standard combining treatment group [(76.2±14.3) ml· min-1 · 1.73 m-2 vs.(71.9±17.9) ml· min-1 · 1.73 m-2,P＜0.05],while Scr,eGFR and uNGAL showed no changes in intensively combining treatment group and intensive atorvastatin therapy group (P＞0.05) ; BUN in the two groups was decreased [(5.6± 1.4)mmol/L vs.(4.7±0.9) mmol/L,(5.3±1.2) mmol/L vs.(4.8±1.2) mmol/L,P＜0.01,P＜0.05].SUA was reduced in intensively combining treatment group (P ＜ 0.05).uNGAL was increased in standard combining treatment group (P ＜ 0.05).Conclusions For elderly patients,intensive atorvastatin therapy and combining intensive treatment can both improve CIAKI.Only combination and intensive treatment benefit for decrease of uric acid.

Objective:To quantitatively evaluate the guidelines for hypertensive disorders of pregnancy (HDP) at home and abroad, and to provide a reference for the development of related guidelines in the future.Methods:Guidelines related to HDP published at home and abroad from 1 January 2018 to 31 December 2022 were retrieved from several databases, including CNKI, Wanfang Database, Yiigle, VIP Database, PubMed, Embase, and Web of Science with the terms of "hypertension in pregnancy", "hypertensive disorders of pregnancy", "pre-eclampsia", "eclampsia", and "guidelines". The retrieved guidelines were evaluated with the Appraisal of Guidelines for Research and Evaluation Ⅱ (AGREE Ⅱ) and Scientific, Transparent and Applicable Rankings (STAR) tool. According to the manual of AGREE Ⅱ two researchers graded the retrieved guidelines from six domains: scope and purpose, participants, rigor, clarity, applicability, and independence. Mean standardized score of each domain and the overall score were obtained. STAR tool was used to grade the guidelines by two researchers and one methodologist from 11 domains: registration, protocol, funding, working groups, conflicts of interest, clinical issues, evidence, consensus methods, recommendations, accessibility, and others.Results:A total of 19 related guidelines were included, covering six countries on three continents. The mean standardized scores of the 19 guidelines in the six domains of scope and purpose, participants, rigor, clarity, applicability, and independence using the AGREE II instrument were 73.98%, 63.16%, 59.98%, 66.37%, 56.36%, and 71.93%, respectively. Scores in the 11 domains of registration, protocol, funding, working groups, conflicts of interest, clinical issues, evidence, consensus methods, recommendations, accessibility, and others using the STAR tool were 0.00%, 0.00%, 76.15%, 39.87%, 58.92%, 65.19%, 60.80%, 49.78%, 78.95%, 30.89%, and 42.11%, respectively. According to the overall evaluation results, 12 guidelines were recommended and seven needed further modifications. It was found that most guidelines were unanimous in their recommendations on the prevention of preeclampsia with aspirin, medications for patients with severe hypertension, and the timing of pregnancy termination in preeclampsia patients, with the consensus rates of 10/13, 9/13, and 9/13, respectively. Besides, these recommendations were supported by substantial evidence.Conclusions:The overall quality of guidelines related to HDP at home and abroad is high, but there is still room for improvement. When developing relevant guidelines in the future, statisticians and methodologists should be included in the working groups to improve the evidence-based quality, and much attention should be paid to the disclosure of conflicts of interest guidelines. Registration and protocol are needed before publishing a guideline. The development of multiple versions for different users will conduce to improving the management of HDP.

Objective To evaluate the safety,feasibility of the application of "four-quadrant" tumor target-artery positioning in "zero-ischemia" retroperitoneal laparoscopic partial nephrectomy.Methods A retrospective study was conducted on 25 patients who underwent retroperitoneal laparoscopic partial nephrectomy from November 2016 to March 2017 by single surgeon.There were 15 males and 10 females with a mean age of (52.6 ± 11.9) years old.The mean diameter of the tumor was (3.8 ± 0.5) cm.The mean R.E.N.A.L.score was (9.0±1.3).The mean GFR of the operation side was (49.2±11.4) ml/(min · 1.73 m2) before the operation.All 25 patients underwent 3 Dimentional CT for the reconstruction of their renal models.The tumor target-artery was located in "four-quadrant" system,which based on the boundary between tumor and renal parenchyma.Perioperative outcomes including the estimated blood loss (EBL),operation time,complications and oncological outcomes were carefully collected and analyzed.Results Of all the 25 patients,21 patients underwent "zero-ischemia" retroperitoneal laparoscopic partial nephrectomy,the mean operation time was (106.1 ± 18.8) min,the mean EBL was (162.2 ±68.0) ml with no transfusion case.4 patients converted to conventional retroperitoneal laparoscopic partial nephrectomy,the operation time was 110-140 min,the EBL was 350-500 ml and the warm-ischemia time was 12-20 min respectively.The pathological outcome shows renal cell carcinoma in 24 patients and papillary renal cell carcinoma in 1 patient with all negative margins.The mean GFR of the operation side was (45.1 ± 10.2) ml/(min · 1.73 m2) after three months of the surgery and shows no statistical difference (P =0.268).Conclusion The application of the "four-quadrant" tumor target-artery positioning in " zeroischemia" retroperitoneal laparoscopic partial nephrectomy is a safety and feasible method in terms of accurate location of the target-artery,better postoperative renal function,low EBL and reasonable oncological outcomes.

Objective To evaluate the efficacy of modified abdominal laparoscopy in the repair of complex vesical vaginal fistula after total hysterectomy.Methods The clinical data of 58 cases of urinary bladder and vagina fistula in our hospital from April 2014 to December 2017 were retrospectively analyzed,of which 32 cases were repaired by ordinary abdominal laparoscopy from April 2014 to February 2016 and 26 cases were repaired by modified abdominal laparoscopy from March 2016 to June 2017.On the basis of the original laparoscopy,the modified transabdominal laparoscopy enlarged the free range between the vaginal stump and the bladder,separated the anterior vaginal wall from the bladder completely and dissociated the retrovaginal peritoneum,wrapping around the vaginal stump to make the peritoneum.The median age of ordinary group was 52 (range:33-67)years old,the median course of disease was 12 (range:3-40) months,and the size of fistula was (25.5 ± 10.3) mm.The median age of modified group was 50 (range:37-65) years,the median course of disease was 11.5 (range:3-36) months,and the size of the fistula was (26.3 ± 9.1) mm.The operation time,bleeding volume,the time of hospitalization,the rate of complications and the success rate of the operation were compared.Results The operation time of the two groups was successfully completed.The operation time of the modified group [(164.2 ± 21.2) min] was significantly shorter than that of the common group [(201.4 ± 25.8) min],and the difference was statistically significant (P ＜ 0.01).In the modified group,the cure rate (100.0％,26/26) was higher than that in the normal group (84.4％,27/32,P ＜ 0.05).There was no significant difference in the amount of intraoperative bleeding[50 (10-100) ml vs.55 (5-110) ml],hospitalization time [(9.1 ± 1.7) d vs.(10.0 ± 1.8) d],postoperative infection [19.2％ (5/26) vs.15.6％ (5/32)],urinary incontinence [7.7％ (2/26) vs.9.4％ (3/32)],urinary frequency [15.4％ (4/26) vs.21.9％ (7/32)],intestinal obstruction [3.8％ (1/26) vs.9.4％ (3/32)] between the modified group and common group (P ＞ 0.05).Conclusions The modified transabdominal laparoscopic mode shortens the operation time,and improves the cure rate of the operation.Satisfactory results are recommended for the repair of high complex bladder vagina fistula.