Objective:To explore the efficient construction of HSF1 gene knockout mouse model using CRISPR/Cas9 gene editing technology, and to establish the early basis for the mouse model of primary osteosarcoma.Methods:According to exon 9 of HSF1 gene structure, the corresponding GRNA (guideRNA) was selected and screened. Then the transcription template of sgRNA (small guide RNA) was amplified by PCR, and four up stream primers were obtained. Subsequently, sgRNA was transcribed in vitro and screened by Tube Screen platform to screen the sgRNA with effective cutting, and the sgRNA with the highest cutting efficiency was selected from the screening results for subsequent experiments. The transcription template of SPCas9mRNA was amplified by PCR, and then Cas9mRNA was transcribed in vitro. The sgRNA transcribed in vitro and Cas9mRNA were injected into the fertilized eggs of healthy C57BL/6 mice, and the tissue was extracted from the tail of the born mice and identified by PCR sequencing. Heterozygous female mice of F0 generation were selected to mate with wild-type male mice too btain F1 generation off spring. The mutation of gene bases of F1 generation mice was detected by AGAR gel electrophoresis and gene sequencing. The heterozygous male mice of the F1 generation and female mice of the F0 generation were back crossed to obtain the F2 generation daughter mice. The tail tissues were cut and sequenced to obtain the F2 generation homozygous knockout mice. PCR was used to observe the cutting efficiency of sgRNA and the sequencing of rat tail tissue, and SNAPGene software was used for gene sequence alignment to determine the deletion of base fragments.Results:The up stream primers sgRNA-1 Primer-f, sgRNA-2 Primer-f, sgRNA-3 Primer-f, sgRNA-4 Primer-f and down stream primers sgRNA-4 Primer -r were obtained by PCR amplification. After in vitro tran scription and screening of sgrRNA, sgrRNA-1, sgrRNA-2 and sgrRNA-4 had high cleavage efficiency and were selected for subsequent experiments. T7 promoter was added to the 5 'end of Cas9 mRNA, and Cas9 mRNA was obtained by PCR and in vitro transcription kit. Mixed Cas9-sgRNA solution was injected into the fertilized eggs of mice and cultured. The cultured two-cell fertilized eggs were injected into the ampulla of the pseudo pregnant female mice, and the F0 generation mice were obtained successfully. A total of 8 heterozygous mice of F0 generation were obtained by Agar gel electrophoresis. Three heterozygous knockout mice of F1 generation were obtained by breeding the female heterozygous mice of F0 generation with healthy wild-type male mice and PCR and sequencing. Three heterozygous male mice of F1 generation were back crossed with female mice of F0 generation 3 to obtain F2 generation mice. Through the observation of electrophoresis and sequencing results of F2 generation mice, it was confirmed that 7 mice were missing HSF1 base sequence, and the electrophoresis results showed mutant bands and no wild-type bands, which were identified as homozygous. The F2 generation homozygous mice were able to breed stably. As eries of results proved that the HSF1 gene knockout mouse model was successfully established in this experiment.Conclusion:CRISPR/Cas9 technology was successfully used to construct HSF1 gene knockout mouse model, with strong stability and high reproducibility, which laida foundation for further study of HSF1 gene expression products and establishment of mouse model of primary osteosarcoma.

Objective:To compare ultrasound (US) guided versus computed tomography (CT) guided radiofrequency ablation (RFA) in treatment of early hepatocellular carcinoma (HCC).Methods:The data of 133 patients with early HCC treated by RFA in the Department of Hepatobiliary Surgery of Shandong Provincial Hospital from February 1, 2015, to January 31, 2017, was analyzed retrospectively. These patients were divided into two groups: the US-guided group and the CT-guided group. The clinical data was collected and the factors affecting prognosis were analyzed.Results:Compared with the CT-guided group, the operation time of the US-guided group was significantly shorter [(29.0±12.0)min vs. (55.0±19.0)min, P<0.05], but the number of ablation sessions per tumor was significantly less [(1.1±0.3) vs. (2.0±0.6), P<0.05]. There was no significant difference in the complete ablation rates, postoperative complication rates and postoperative length of hospital stay between the two groups ( P>0.05). The CT-guided group was superior to the US-guided group in the local tumor recurrence and progression-free survival rates ( P<0.05). On multivariate analysis, CT-guided RFA was an independent protective factor for local tumor recurrence ( HR=0.266, 95% CI: 0.073-0.967, P<0.05) and progression-free survival ( HR=0.415. 95% CI: 0.213-0.806, P<0.05), while AFP >20 ng/ml ( HR=4.821, 95% CI: 1.714-13.560, P<0.05) was an independent risk factor for progression-free survival. Conclusion:CT-guided percutaneous RFA was superior to US-guided RFA in local treatment of early HCC, probably related to more needle placements and longer ablation time under CT guidance.

Hepatolithiasis is a common biliary tract disease in China and other Asian countries.It has insidious onset,recurrent disease,and may lead to biliary cirrhosis or even cancer in the late stage without inter-vention.At present,surgery is considered to be the best choice for treatment.In the 21st century of minimally inva-sive surgery,traditional procedures such as partial hepatectomy,choledochotomy,cholangioenterostomy and liver transplantation can be performed with the assistance of laparoscopy and da Vinci robots,and emerging endoscopic techniques such as endoscopic retrograde cholangiopancreatography(ERCP)and percutaneous transhepatic cho-ledochoscopic lithotripsy(PTCSL)have also been developed,bringing better treatment for patients with hepatoli-thiasis.As treatment options are becoming more varied,there are also some clinical problems that need to be addressed.In this article,we would like to briefly review the current surgical treatment modalities in order to provide a theoretical basis for optimizing the treatment modalities.

Objective:To investigate the efficacy and safety of bladder neck resection combined with multipoint injection of triamcinolone acetonide in the treatment of bladder neck contractures (BNC) after transurethral resection prostate (TURP).Methods:The data of 25 patients with BNC after TUPR who underwent transurethral surgery in West China Hospital of Sichuan University from July 2019 to November 2021 were retrospectively analyzed, and the patients were divided into 2 groups according to the treatment method. There were 15 cases in the steroid injection group, with an average age of (67.5±8.8) years, 1 case of diabetes, and 2 cases of hypertension. And the average postoperative time after TURP was (21.9±29.1) months, the preoperative International Prostate Symptom Score (IPSS) was (30.0±3.5) points, quality of life (QOL) score was (5.7±0.5) points. There were 10 cases in the non-steroid injection group, with an average age of (65.2±10.5) years, 1 case of diabetes, and 2 cases of hypertension. And the average postoperative time of TURP was (29.3±33.5) months, and the preoperative IPSS was (30.4±2.6) points, QOL score was (5.8±0.4) points. There was no significant difference between the two groups ( P>0.05). In the steroid injection group, bladder neck resection combined with multipoint injection of triamcinolone acetonide was performed. The patients were in the lithotomy position, and a scope was placed through the urethra into the distal end of the narrow urethra under direct vision for observation, and a supersmooth guide wire was placed. The narrow section is then incised at 6 o'clock. After replacing the resectoscope, the urethral stricture scar was excised until the normal tissue of the bladder neck was exposed. Hemostasis by electrocoagulation resulted in no active bleeding from the urethra after resection. After the bladder injection needle was inserted, 80 mg (12 ml) of triamcinolone acetonide injection was injected in 6 equally spaced needles at the direction of the bladder neck from 3 to 9 o'clock. A supersmooth guide wire was placed, and a three-channel silicone urinary catheter was indwelled along the guide wire. The non-steroid injection group underwent simple bladder neck resection. Re-examination of urethroscopy at 3 and 6 months after operation showed that the patient had obvious dysuria and the microscopic examination showed that the recurrence of bladder neck stenosis was defined as the recurrence of stenosis. The efficacy, complications, and recurrence-free survival rates of the two groups were compared. Predictors of postoperative BNC recurrence were analyzed. Results:The operations in both groups were successfully completed. The operation time of the steroid injection group and the non-steroid injection group were (36.0±17.8) min and (48.5±57.9) min respectively ( P=0.438), and the intraoperative blood loss was (1.9±3.0) ml and (12.0±31.1) ml ( P=0.221)respectively. The length of hospital stay was (5.8±1.2) d and (4.4±2.5) d, respectively ( P=0.070). There was 1 case of transient hematuria and 1 case of epididymitis in the steroid injection group, and 1 case of transient hematuria and 1 case of transient dysuria in the non-steroid injection group ( P>0.05), all of which were relieved after symptomatic treatment. The IPSS of steroid injection group and non-steroid injection group were (11.0±5.6) points and (12.4±3.9) points at 3 months after operation, and (10.1±4.9) points and (14.7±7.7) points at 6 months after operation, respectively. QOL at 3 months after operation was (1.7±1.2) points and (2.1±1.5) points, respectively, and at 6 months after operation, it was (1.5±1.3) points and (3.0±2.0) points, respectively. There was statistical significance ( P<0.05). There were 1 case and 2 cases of recurrence in the steroid injection group and non-steroid injection group at 3 months after operation ( P=0.543), and 1 case and 5 cases at 6 months after operation ( P=0.023). The difference in survival curve between the two groups was statistically significant ( P=0.013). Combined steroid therapy ( OR=14.000, 95% CI1.299-150.889, P=0.030), time after scar resection ( OR=1.138, 95% CI1.017-1.273, P=0.025), postoperative IPSS( OR=1.302, 95% CI1.018-1.666, P=0.036), postoperative QOL score ( OR=4.280, 95% CI1.523-12.030, P=0.006) were the predictors of stenosis recurrence 6 months after surgery. Conclusion:Bladder neck scar resection combined with steroid injection could be safe and effective in the treatment of BNC after TURP, and local steroid injection may help reduce the recurrence rate of postoperative stenosis.