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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.
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Objective:To investigate the clinical effect of lateral rectus abdominis approach combined with presacral decompression for surgical treatment of old Denis type II sacral fractures complicated with upper sacral plexus injury.Methods:A retrospective case series study was performed on the clinical data of 9 patients with old Denis type II sacral fractures complicated with upper sacral plexus injury (L 4-S 1) admitted to Zhongnan Hospital of Wuhan University from June 2010 to December 2016. There were 6 males and 3 females, aged (33.1±7.5)years (range, 15-58 years). Embolization of internal iliac artery and preimplantation of abdominal aortic balloon were performed 2 hours before operation under the guidance of digital subtraction angiography (DSA). Surgery was performed using a single lateral rectus abdominis approach combined with presacral decompression. The operation time, intraoperative blood loss and full weight-bearing time were recorded. The visual analogue scale (VAS) and European QOL Five Dimensional health scale (EQ-5D) were compared before and after operation. The Gibbons' impairment scale was used to assess neurological function. X-ray and CT scan were used to observe internal fixation and fracture healing. The complications during and after operation were recorded. Results:The patients were followed up for 24-52 months [(35.2±5.2)months]. The operation time was (2.9±0.6)hours. The intraoperative bleeding was (573±138)ml, and the full weight-bearing time was (11.6±1.2)weeks. X-ray and CT scan showed bone healing in all patients at the latest follow-up. The VAS and EQ-5D scale improved from preoperative (7.8±0.6)points and (0.34±0.07)points to the final follow-up of (0.8±0.3)points and (0.81±0.05)points ( P<0.05). According to Gibbons classification, 8 patients were grade I and 1 patient was grade II one year after operation ( P<0.01). Namely, the radiation pain of lower extremities was significantly improved in all patients, among which 8 patients showed pain disappeared and completely returned to normal and 1 patient showed residual numbness and hypoesthesia of the affected limbs. No major complications (eg, iatrogenic lumbosacral plexus injury, vital blood vessels or pelvic organs injury) occurred during the operation. During the follow-up period, only one patient developed traumatic hip arthritis and underwent total hip arthroplasty 6 months after operation. Fractures of the remaining patients were healed well without complications like infection, pressure ulcer or implant failure. Conclusions:For old Denis type II sacral fractures complicated with upper sacral plexus injury, lateral rectus abdominis approach combined with presacral decompression can fully decompress the upper sacral plexus nerve, relieve pain, and promote functional rehabilitation, with low incidence of complications. It is an alternative surgical method for the treatment of old Denis type II sacral fractures complicated with upper sacral plexus injury.
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Objective To compare the lateral rectus abdominis approach versus posteromedian sacrum approach in the surgical treatment of unstable sacral fracture complicated with lumbosacral plexus injury.Methods From June 2010 to December 2014,33 unstable sacral fractures complicated with lumbosacral plexus injury were treated at Department of Orthopaedics,Spinal and Pelvic Surgery Center,Zhongnan Hospital.Of them,24 were fresh and 9 obsolete.The lateral rectus abdominis approach was adopted in 17 patients and the posteromedian sacrum approach in 16.All the patients received surgical treatment within 2 to 12 weeks (average,4.5 weeks) after injury.The 2 groups were compared in terms of operation time,bleeding volume,complications,weight-bearing time,visual analogue scale (VAS),European QOL Five Dimensional health scale (EQ-5D) and lumbosacral injury classification and severity scale (LSICS).Results The 2 groups were compatible due to their insignificant differences in baseline data (P > 0.05).Their follow-up time ranged from 17 to 37 months (average,26.8 months).Perioperatively,infection and necrosis of the lumbosacral incision appeared in 2 patients and iatrogenic injury to the lumbosacral plexus in 3 patients.All the other incisions healed well without major complications like infection,pressure ulcer or implant failure.There were no significant differences between the 2 groups in operation time (235.8± 72.0 minutes versus 318.0 ± 64.7 minutes),intraoperative bleeding volume (558.8 ± 125.7 mL versus 734.0 ±98.0 mL),weight-bearing time (9.4 ±2.4 weeks versus 11.3 ±2.3 weeks),postoperative complications,VAS(1.1 ±0.6 points versus 1.0 ±0.6 points),EQ-5D (0.82 ±0.09 points versus 0.78 ±0.06 points) or LSICS (P > 0.05).The final follow-ups revealed significant improvements in VAS,EQ-5D and LSICS in all the patients (P < 0.05).Conclusion The lumbosacral plexus can be well decompressed via both the lateral rectus abdominis approach and the posteromedian sacrum approach,leading to satisfactory clinical outcomes.
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Objective To investigate the surgical treatment of complex Denis zone Ⅲ sacral fractures.Methods A retrospective study was conducted on the 15 complex Denis zone Ⅲ sacral fractures which had been treated from June 2011 to September 2015 in our hospital.They involved 9 men and 6 women,with an average age of 28.8 years (from 15 to 55 years).There were 8 U-shaped,6 H-shaped and 3 lambda fractures.By the Gibbons grading for the sacral nerve injury,3 cases were rated preoperatively as grade Ⅱ,5 cases as grade Ⅲ and 7 as grade Ⅳ.Of them,10 received π-shaped lumbopelvic fixation and sacral decompression 2 weeks after injury,one had lumbosacral fixation and sacral decompression 6 days after injury,and 4 underwent only sacral decompression 3 months after injury when they presented with symptoms of neural injury after fracture malunion.Their sacral malformation angle,visual analogue scale and EQ-5d scale were compared between preoperation and postoperation.Results The 15 cases were followed up for 9 to 47 months (average,24.9 months).Their full-weight-bearing time ranged from 8 to 14 weeks (average,9.9 months).All the incisions healed well without any infection,pressure sore,implant loosening or breakage.There was no significant difference between the preoperative angle of malformation (35.7° ± 5.0°,from 15° to 90°) and the postoperative value (27.9°±6.0°,from 8° to 90°) (t =1.003,P=0.324).Their VAS pain score decreased significantly from preoperative 7.0 ±0.3 to postoperative 1.9 ±0.2,and their EQ-5D score increased significantly from preoperative 0.20 ± 0.03 to postoperative 0.79 ± 0.04 (P < 0.05).The final follow-ups revealed more or less recovery of neural function in all the patients.Conclusions π-shaped lumbopelivic fixation is an effective treatment for complex Denis zone Ⅲ sacral fractures.Effective decompression and stable fixation are prerequisites for early neurological and functional recovery.Simple sacral decompression can effectively promote nerve recovery in patients with old complex Denis zone Ⅲ sacral fractures.
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BACKGROUND:Early-onset scoliosis is a kind of disease that seriously affects the growth of children’s spine and development of cardiorespiratory function. The treatment of the disease has always been the focus of many clinical researchers.OBJECTIVE:To analyze the therapy for early-onset scoliosis and explore the spinal fusion, spinal non-fusion, conventional growth rod technology and magnetic control ed growth rod technology of early-onset scoliosis. METHODS:We retrieved PubMed, CENTRAL, EMbase, the ISI Web of Knowledge Databases, VIP, CNKI, CBM and Wanfang Database for related studies published from inception of the database to March 2016. The key words were“scoliosis, growing rod, complications”. The included 54 studies were analyzed and discussed. RESULTS AND CONCLUSION:For these children of early-onset scoliosis, we should not only maintain the correction of spine deformities, but also protect the ability of spine growth, keeping the normal cardiopulmonary function. In addition to conventional (non-surgical) treatment, there are surgical treatment (such as spinal fusion and growing rod technique) and magnetical y control ed growing rod, a new technology for the treatment of early-onset scoliosis. A comprehensive understanding of the effect of surgical treatment on the spine growth and cardiopulmonary function of children with early-onset scoliosis wil help to prevent the occurrence of related complications, so as to obtain a better therapeutic effect.
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Treatment of calcaneal fractures has always been the focus of many clinical researchers.The goals of traditional surgical treatment are not only to restore the integrity of calcaneal articular surface but also to reconstruct the anatomy of the calcaneus.More importantly,we need to reduce postoperative soft tissue swelling and incidence of postoperative complications.In recent years,scholars have reported satisfactory clinical efficacy and prognosis resulting from a sinus tarsal approach for treatment of calcaneal fractures.This paper reviews the latest research progress concerning the sinus tarsal approach for treatment of calcaneal fractures at home and abroad,intending to provide helpful information for the clinical surgeons.
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BACKGROUND:Supination maneuver is mainly used for reduction of radial head subluxation in children, but recently, pronation maneuver has also achieved good results in the treatment of radial head subluxation. OBJECTIVE:To objectively evaluate the efficacy of pronationversus supination maneuvers for the reduction of radial head subluxation by using Meta-analysis. METHODS:PubMed, Cochrane Central Register of Controled Trials (CENTRAL), EMbase, the ISI Web of Knowledge databases, VIP, CNKI, CBM and Wanfang were searched from database establishment to December 2014 for colecting the randomized controled trials (RCTs) about pronationversus supination maneuvers for the reduction of radial head subluxation, and the references of those RCTs were also searched by hand. After study selection, assessment and data extraction were conducted by two reviewers independently. Meta-analyses were performed by using the RevMan 5.2 software. RESULTS AND CONCLUSION:Five studies involving 436 patients were included. The results of Meta-analyses showed that: compared with the supination maneuvers group the pronation maneuvers group had a higher rate of successful reduction at the first attempt [RR=1.17, 95%CI (1.08, 1.28),P=0.000 3] and lower rate of failed reduction [RR=0.25, 95%CI(0.09, 0.65),P=0.005]. There was no significant difference in the rate of successful reduction at the second attempt [RR=1.39, 95%CI (0.75, 2.58),P=0.30]. Based on the results of systemic assessment, the level of evidence assessed by the GRADE system showed that the outcome indicators of the rate of successful reduction at the first attempt and rate of failed reduction were graded as intermediate level; the outcome indicator of the rate of successful reduction at the second attempt was graded as low level. For the poor quality of the original studies, a prudent choice is suggested; and more highly-quality, large-sample studies are needed.
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BACKGROUND:A large number of studies have confirmed that minimaly invasive and open transforaminal lumbar interbody fusion approaches for single-segment and multi-segment lumbar degenerative diseases were effective, but their advantages and disadvantages remain inconclusive. OBJECTIVE:To systematicaly evaluate the effectiveness and safety of minimaly invasive versus open transforaminal lumbar interbody fusion for lumbar degenerative disease. METHODS: Databases including The Cochrane Library (Issue 2, 2014), PubMed, EMbase, MEDLINE, SCI, CNKI, CBM, Wanfang Data were searched to colect the randomized controled trails and non-randomized controled trails about minimaly invasive and open transforaminal lumbar interbody fusion for lumbar degenerative disease. The retrieval time was from foundation to February 2014. The studies were screened according to inclusion and exclusion criteria. The data were extracted and the quality was evaluated by two reviewers independently. Meta-analysis was conducted using RevMan 5.2 software. RESULTS AND CONCLUSION:A total of 1 randomized controled trail and 18 non-randomized controled trails involving 1 400 patients were included. The results of meta-analysis showed that: compared with open transforaminal lumbar interbody fusion, minimaly invasive transforaminal lumbar interbody fusion had many advantages, such as smal paraspinal muscle injury, less blood loss, short hospital stay, good early effects and high long-period remission rate of lumbago. However, minimaly invasive transforaminal lumbar interbody fusion was not superior to open transforaminal lumbar interbody fusion in improving long-period motor function, long-term remission rate of leg pain and in reducing complications. Moreover, the operation time of minimaly invasive transforaminal lumbar interbody fusion was longer. Therefore, under the premise of strict indications, minimaly invasive transforaminal lumbar interbody fusion could better solve the complaint of patients with lumbar degenerative disease. For the quantitative and qualitative limitation of the included studies, this conclusion stil requires to be further proved by performing a larger scale and a higher quality of randomized controled trails. It suggests that doctors should choose a best therapy for patients according to an integrative disease assessment.