1.Evidence-based clinical practice guideline for bone cement-augmented pedicle screw technique (version 2025)
Sihao HE ; Junchao XING ; Tongwei CHU ; Zhengqi CHANG ; Xigao CHENG ; Fei DAI ; Xiaobing JIANG ; Jie HAO ; Jiang HU ; Jinghui HUANG ; Tianyong HOU ; Fei LUO ; Bo LIAO ; Changqing LI ; Lei LIU ; Guodong LIU ; Peng LIU ; Sheng LU ; Weishi LI ; Yang LIU ; Zhen LIU ; Wei MEI ; Peifu TANG ; Bing WANG ; Bing WANG ; Ce WANG ; Hongli WANG ; Liang WANG ; Shengru WANG ; Xiaobin WANG ; Yang WANG ; Yingfeng WANG ; Zheng WANG ; Jianzhong XU ; Guoyong YIN ; Haiyang YU ; Qiang YANG ; Zhaoming YE ; Bin ZHANG ; Chengmin ZHANG ; Jun ZOU ; Qiang ZHOU ; Min ZHAO ; Rui ZHOU ; Xiaojun ZHANG ; Yongfei ZHAO ; Zhongrong ZHANG ; Zehua ZHANG ; Yingze ZHANG
Chinese Journal of Trauma 2025;41(11):1035-1047
For middle-aged and elderly patients with conditions such as spinal fractures and degenerative spinal diseases, spinal internal fixation is a core surgical procedure for reconstructing spinal stability, heavily relying on the biomechanical stability provided by pedicle screw systems. Whereas, these patients are often complicated by osteoporosis that can significantly compromise the stability of the bone-pedicle screw interface, leading to a marked increase in pedicle screw loosening and surgical failure rates. The bone cement-augmented pedicle screw technique, which involves injecting bone cement into the vertebral body or screw trajectory to optimize the mechanical properties of the bone-pedicle screw composite, has been proven to significantly enhance fixation strength and effectively prevent screw-related failures, thereby reducing the incidence of internal fixation failure in high-risk populations undergoing spinal fusion. However, the widespread clinical application of this technique has faced challenges such as inaccurate clinical decision-making (indication and contraindication selection), non-standardized operative practices, and insufficient awareness of complication prevention, resulting in considerable variability in clinical outcomes and even severe complications. To address this, Prof. Luo Fei from First Affiliated Hospital of Army Medical University initiated the project and the Chinese Association Orthopaedic Surgeons organized relevant experts to develop the Evidence-based clinical practice guideline for bone cement-augmented pedicle screw technique ( version 2025), based on current evidence. The guidelines put forward 8 recommendations regarding the clinical value, scope of application, and operational standards of the technique, aiming to provide evidence-based medical support and technical standardization for clinical decision-making.
2.Evidence-based clinical practice guideline for bone cement-augmented pedicle screw technique (version 2025)
Sihao HE ; Junchao XING ; Tongwei CHU ; Zhengqi CHANG ; Xigao CHENG ; Fei DAI ; Xiaobing JIANG ; Jie HAO ; Jiang HU ; Jinghui HUANG ; Tianyong HOU ; Fei LUO ; Bo LIAO ; Changqing LI ; Lei LIU ; Guodong LIU ; Peng LIU ; Sheng LU ; Weishi LI ; Yang LIU ; Zhen LIU ; Wei MEI ; Peifu TANG ; Bing WANG ; Bing WANG ; Ce WANG ; Hongli WANG ; Liang WANG ; Shengru WANG ; Xiaobin WANG ; Yang WANG ; Yingfeng WANG ; Zheng WANG ; Jianzhong XU ; Guoyong YIN ; Haiyang YU ; Qiang YANG ; Zhaoming YE ; Bin ZHANG ; Chengmin ZHANG ; Jun ZOU ; Qiang ZHOU ; Min ZHAO ; Rui ZHOU ; Xiaojun ZHANG ; Yongfei ZHAO ; Zhongrong ZHANG ; Zehua ZHANG ; Yingze ZHANG
Chinese Journal of Trauma 2025;41(11):1035-1047
For middle-aged and elderly patients with conditions such as spinal fractures and degenerative spinal diseases, spinal internal fixation is a core surgical procedure for reconstructing spinal stability, heavily relying on the biomechanical stability provided by pedicle screw systems. Whereas, these patients are often complicated by osteoporosis that can significantly compromise the stability of the bone-pedicle screw interface, leading to a marked increase in pedicle screw loosening and surgical failure rates. The bone cement-augmented pedicle screw technique, which involves injecting bone cement into the vertebral body or screw trajectory to optimize the mechanical properties of the bone-pedicle screw composite, has been proven to significantly enhance fixation strength and effectively prevent screw-related failures, thereby reducing the incidence of internal fixation failure in high-risk populations undergoing spinal fusion. However, the widespread clinical application of this technique has faced challenges such as inaccurate clinical decision-making (indication and contraindication selection), non-standardized operative practices, and insufficient awareness of complication prevention, resulting in considerable variability in clinical outcomes and even severe complications. To address this, Prof. Luo Fei from First Affiliated Hospital of Army Medical University initiated the project and the Chinese Association Orthopaedic Surgeons organized relevant experts to develop the Evidence-based clinical practice guideline for bone cement-augmented pedicle screw technique ( version 2025), based on current evidence. The guidelines put forward 8 recommendations regarding the clinical value, scope of application, and operational standards of the technique, aiming to provide evidence-based medical support and technical standardization for clinical decision-making.
3.Effects of pharyngeal cavity and mentolingual muscle exercise on polysomnography,cardiac function and MACE in patients with mild OSAHS left after palatopharyngoplasty with diastolic cardiac dysfunction
Sheng LI ; Mang XIAO ; Xiaohua JIANG ; Qingye YANG ; Jinshan LAN ; Hongjian LIAO ; Hongwei ZHANG ; Yin FU
Chinese Archives of Otolaryngology-Head and Neck Surgery 2024;31(7):461-466
OBJECTIVE To investigate the effect of pharyngeal cavity and genoglossus muscle exercises in patients with mild obstructive sleep apnea syndrome(OSAHS)left over after palatopharyngoplasty with diastolic cardiac dysfunction.METHODS A total of 75 patients with mild OSAHS left after palatopharyngoplasty with diastolic cardiac dysfunction from January 2021 to August 2023 were selected for retrospective study.Among them,37 patients underwent pharyngeal cavity and mentoglossum muscle exercise(observation group),while 38 patients did not(control group).Using postoperative data as baseline value.mean blood oxygen saturation(MSpO2),lowest oxygen saturation(LSpO2),sleep efficiency,apnea index(AI),sleep latency,apnea hypopnea index(AHI),hypopnea index(HI),microarousal index(MAI),rapid eye movement latency,arterial blood pressure of carbon dioxide(PaCO2),pH,arterial partial oxygen pressure(PaO2),left ventricular end-systolic diameter(LVDs),blood lactic acid,left ventricular ejection fraction(LVEF),Tei index,left ventricular end-diastolic diameter(LVDd),daytime Epworth sleepiness scale(ESS)score,Pittsburgh sleep quality index(PSQI)score and incidence of adverse cardiovascular events(MACE)were compared at baseline and 3 months later.RESULTS After 3 months,AHI,HI and AI in observation group were lower than those in control group,and MSpO2 and LSpO2 were higher than those in control group(P<0.05);Sleep efficiency of observation group was higher than control group,daytime ESS score,PSQI score and MAI were lower than control group(P<0.05).There were no significant differences in PaCO2,LVDd,PaO2,blood lactic acid,sleep latency,pH,LVEF,rapid eye movement latency,LVDd,Tei index between the observation group and the control group(P>0.05).The incidence of MACE in the observation group was 5.41%(2/37),compared with 13.16%(5/38)in the control group,there was no significant difference(P>0.05).CONCLUSION In patients with mild OSAHS left after palatopharyngoplasty with diastolic cardiac dysfunction,the exercise of pharyngeal cavity and genoglossus can improve hypopnea,alleviate clinical symptoms and improve sleep quality,but it has limited effect on the improvement of cardiac function.
4.The effect of bulk electron density on the dose accuracy of treatment planning for postoperative cervical cancer
Sheng HAN ; Xiaodong PENG ; Ke YUAN ; Rutie YIN ; Xiongfei LIAO ; Xianliang WANG ; Lei ZHANG ; Min LIU ; Bin BI ; Yazheng CHEN
Chinese Journal of Radiological Medicine and Protection 2021;41(2):140-145
Objective:To study the impact on dose accuracy for the treatment planning by manually assigning accurate electron density for CT image-based tumor tissues and organs at risk.Methods:Twenty cases of retrospective postoperative cervical cancer radiotherapy plans were selected. The body electron density of the corresponding organs was derived from the ICRU 46 report and assigned in the treatment planning system (Monaco5.11, Sweden), including the bladder, rectum, intestine, kidney, spinal cord, femoral head, and ilium. The original plans were double-arc volumetric modulated arc therapy plan (360° VMAT), using Monte Carlo algorithm, the calculation grid was 0.3 cm × 0.3 cm × 0.3 cm, and the minimum subfield width was 0.6 cm. Keep the original plan fluence unchanged and recalculate the dose to generate a new plan. The two-dimensional dose distribution and dose-volume histogram (DVH) were used to compare the differences between the two plans. The difference was compared between the two group plans by using the dosimetry parameters and DVH two dimension curve.Results:For the planning of assigning bulk electron density (Plan RED), the deviation of the patient′s target dose parameters and the original plan (Plan ref) was <2%, and the average deviation of all target regions D2, D98, Dmean was < 0.7%, only 2 of the 180 data were between 2% and 3%. The average deviation of V20, V30, D1 cm 3, Dmean of the bladder, rectum, and small intestine, the original Plan ref was less than 0.6%, and 4 out of 240 data had values > 2%. Plan RED′s average hop count was 0.9% higher than Plan ref, and the total number of subfields remains unchanged. The planned dose generated by manually assigning the electron density in Plan RED was higher than that in Plan ref, but met the clinical requirements. The two-dimensional curves of the DVH diagram for targets and OARs almost completely overlapped, and there was no obvious difference in the dose distribution diagram of the same cross section. The statistical result of all parameters showed that the difference in planned dose parameters between the two groups was not statistically significant( P>0.05). Conclusions:The overall deviation of dose accuracy between Plan RED and Plan ref is <2%, which meets the clinical requirements and provides a reference for realizing MRI-only treatment planning.
5. Application of Chen′s pancreaticojejunostomy technique in laparoscopic pancreaticoduodenectomy (116 cases report)
Xinmin YIN ; Yunfeng LI ; Wei CHENG ; Chunhong LIAO ; Yi LIU ; Yifei WU ; Rongyao CAI ; Siwei ZHU ; Sheng LIU ; Shu WU ; Xiaoping CHEN
Chinese Journal of Surgery 2020;58(2):114-118
Objective:
To investigate the safety and feasibility of longitudinal transpancreatic U-sutures invaginated pancreatojejunostomy (Chen′s pancreaticojejunostomy technique) in laparoscopic pancreaticoduodenectomy (LPD) .
Methods:
Clinical data of 116 consecutive patients who underwent LPD using Chen′s pancreaticojejunostomy technique in Hunan Provincial People′s Hospital from May 2017 to December 2018 were retrospectively analyzed. Among these patients, 66 were males and 50 were females. The median age was 58 years old (32-84 yeas old). All 116 patients underwent pure laparoscopic whipple procedure with Child reconstruction method, using Chen′s pancreaticojejunostomy technique. The intraoperative and postoperative data of patients were analyzed.
Results:
All 116 patients underwent LPD successfully. The mean operative time was (260.3±33.5) minutes (200-620 minutes). The mean time of pancreaticojejunostomy was (18.2±7.6) minutes (14-35 minutes) . The mean time of hepaticojejunostomy was (14.6±6.3) minutes (10-25 minutes). The mean time of gastrojejunostomy was (12.0±5.5) minutes (8-20 minutes). The mean estimated blood loss was (106.0±87.6) ml (20-800 ml). Postoperative complications were: 11.2% (13/116) of cases had postoperative pancreatic fistula (POPF) , including 10.3% (12/116) of biochemical fistula and 0.9% (1/116) of grade B POPF, no grade C POPF occurred; 10.3% (12/116) had gastrojejunal anastomotic bleeding; 3.4% (4/116) had hepaticojejunal anastomotic fistula; 3.4% (4/116) had delayed gastric emptying; 4.3% (5/116) had localized abdominal infection; 12.1% (14/116) had pulmonary infection; postoperative mortality were 0(0/116) and 1.7% (2/116) within 30 days and 90 days, respectively. One patient died of massive abdominal bleeding secondary to Gastroduodenal artery pseudoaneurysm rupture, the other patient died of extensive tumor recurrence and metastasis after surgery.
Conclusions
Chen′s pancreaticojejunostomy technique is safe and feasible for LPD.It is an option especially for surgeons who have not completed the learning curve of LPD.
6.NPM1 High Mutant Allele Burden is an Adverse Prognostic Factor for AML Patients with Mutated NPM1.
Jiang-Xue HOU ; Shu-Juan WANG ; Yan-Fang LIU ; Qian-Qian HAO ; Chong WANG ; Tao LI ; Jun-Jun BAI ; Lin-Xiao LIAO ; Cheng-Yu GUO ; Yin-Yin CHANG ; Meng WANG ; Hui SUN ; Xin-Sheng XIE ; Zhong-Xing JIANG
Journal of Experimental Hematology 2019;27(2):365-372
OBJECTIVE:
To investigate the clinical features, accompanying gene mutation characteristics and prognostic factors of adult patients with acute myeloid leukemia with mutated NPM1 (NPM1AML).
METHODS:
Seventy-three patients with newly diagnosed adult NPM1AML were selected. The mutations of 22 genes were detected by second generation sequencing and 43 fusion genes of AML were detected by real-time fluorescent quantitative PCR. The Kaplan-Meier survival curve and Cox multivariate regression analysis were used to study the prognostic factors.
RESULTS:
A total of 74 NPM1 site mutations were detected in 73 patients with NPM1AML. The incidence rates were 92.0% L287fs, 2.7% Q289fs and W288fs, 1.4% L258fs and Q289H, among which 1 patient had 2 NPM1 mutations; the different mutation sites had no effect on the prognosis of NPM1AML. The median value of NPM1 variant allele frequency (VAF) was 35.4% (1.8%-56.6%). Based on the uppermost quartile of 38.4%, the patients were classified as NPM1 VAF>38.4% (NPM1AML) and NPM1 VAF≤38.4% (NPM1AML). Compared with NPM1AML, the early mortality rate was statistically significantly higher (33.3% vs 7.3%, P<0.05), and median EFS (148 d,95%CI 58-238 d vs 372 d,95%CI 264-480 d) (P<0.01) and median OS (179 d 95%CI 6-352 d vs 444 d) (P<0.01) were significantly shorter in NPM1 AML. A total of 126 accompanying gene mutation sites were detected in 87.7% of patients with NPM1AML. The patients with NRAS gene mutation displayed a higher rate of complete remission (100% vs 58%) (P<0.05) and longer median OS (not reached to 320 d, 95%CI 150-490 d) (P<0.05). The 43 fusion genes were examined in 65 out of 73 cases of NPM1AML, and in all the patients the fusion gene test was negative. Multivariate analysis showed that NPM1 VAF>38.4% was an independent prognostic factor for EFS (HR=3.1, 95% CI 1.6-6.4, P<0.01) and OS (HR=3.0, 95% CI 1.4-6.2, P<0.01).
CONCLUSION
The NPM1 gene mutation in AML patients often is accompanied by other gene mutations, while the coexistence of fusion genes is rare; high NPM1 mutant allele burden is an independent prognostic factor for adult AML patients with mutated NPM1.
Alleles
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Humans
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Leukemia, Myeloid, Acute
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genetics
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Mutation
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Nuclear Proteins
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genetics
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Prognosis
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fms-Like Tyrosine Kinase 3
7.The application of ICG fluorescence imaging in guiding hepatectomy of hepatic carcinoma
Yi LIU ; Xinmin YIN ; Chunhong LIAO ; Yifei WU ; Yunfeng LI ; Sheng LIU
Chinese Journal of Hepatobiliary Surgery 2019;25(2):131-135
Nowadays,surgery is a comprehensive approach for the treatment of hepatic carcinoma as the first choice,but there are still many limitations in surgical therapy,such as the location,boundary and metastasis of hepatic carcinoma.Currently,the indocyanine green fluorescence imaging-guided hepatectomy is widely used at home and abroad as a new progress and hotspot in hepatobiliary surgery,which makes hepatobiliary surgery more convenient and makes up for some deficiencies in hepatectomy.Thus,we summarized the experience of indocyanine green fluorescence imaging-guided hepatectomy in the People's Hospital of Hunan Province and discussed its value of application.
8.Application value of two-step separation approach in laparoscopic hemihepatectomy
Siwei ZHU ; Sheng LIU ; Shu WU ; Xinmin YIN ; Yi LIU ; Wei CHENG ; Chunhong LIAO ; Yifei WU ; Chuang PENG
Chinese Journal of Digestive Surgery 2018;17(5):508-513
Objective To investigate the application value of two-step separation approach in laparoscopic hemihepatectomy.Methods The retrospective cross-sectional study was conducted.The clinicopathological data of 81 patients who underwent laparoscopic hemihepatectomy in the People's Hospital of Hunan Provincial between January 2015 and December 2017 were collected.Patients underwent laparoscopic hemihepatectomy using two-step separation approach after preoperative assessment.Hepatic pedicle,hepatic vein and branches were processed in the liver parenchyma,without intrathecal anatomy.Observation indicators:(1) preoperative assessment,intraand post-operative recovery;(2) follow-up and survival situations.Follow-up using outpatient examination and telephone interview was performed up to February 2018.Patients with hepatolithiasis received abdominal CT reexaminations at 5-7 days postoperatively for observing postoperative residual stones,and patients with malignant tumor were regularly followed up for 1-3 years.Measurement data with normal distribution were represented as (x)±s.Count data were described as frequency and percentage.Results (1) Preoperative assessment,intra-and post-operative recovery:81 patients underwent preoperative blood biochemistry,tumor biomarker and imaging examinations,and remaining functional liver volume and a liver model in 9 patients were respectively calculated and built using the 3D reconstruction software and 3D printing technology.Of 81 patients,68 underwent left hemihepatectomy and 13 underwent fight hemihepatectomy;77 underwent successful laparoscopic hemihepatectomy using two-step separation approach,4 were converted to open operation,with a rate of 4.9% (4/81).Of 4 patients with conversion to open operation,1 had difficult tumor separation due to tumor invading port vein induced to close adhesion,1 had stone removal difficulties under laparoscopy due to hepatolithiasis,and 2 were converted to open operation due to severe liver cirrhosis induced to massive intraoperative bleeding.Of 81 patients,70 gained dividing line of hemiliver by intraoperatively hemihepatic blood flow occlusion,and then got effectively control of bleeding combined with Pringle blood flow occlusion,and 11 received Pringle blood flow occlusion in whole liver.Laparoscopic fluorescence imaging technology was intraoperatively used for 2 patients.Operation time,volume of intraoperative blood loss,rate of intraoperative blood transfusion and duration of hospital stay in 81 patients were respectively (206±42)minutes,(195±134)mL,11.1%(9/81) and (11.5+2.7)days.Eighty-one patients were complicated with bile leakage and were cured by conservative treatment,with a bile leakage incidence of 2.5% (2/81),and without severe complications,such as postoperative bleeding,hepatic dysfunction and subphrenic abscess.There was no perioperative death and reoperation within 30 days postoperatively.(2) Follow-up and survival situations:55 patients with hepatolithiasis were followed up and underwent CT examinations of upper abdomen at 5-7 days postoperatively,including 52 with depletion of stones;3 with residual stones received removal of stones by choledochoscope at 3 months postoperatively,without residual stones.Seventeen patients with malignant tumor were followed up for 12-36 months,with a median time of 15 months,16 had tumor-free survival,and 1 was complicated with intrahepatic metastasis at 1 year after resection of hepatocellular carcinoma,and then underwent transcatheter arterial chemoembolization (TACE) and survived with tumor.Nine patients with benign liver diseases had good recovery during follow-up.Conclusion Two-step separation approach that is rationally used in laparoscopic hemihepatectomy is safe,effective and convenient.
9.Clinical features of patients with bronchiectasis of different types
Weili SHENG ; Yongxiang ZHANG ; Fengxian YIN ; Ying ZHAO ; Yanan LIU ; Tao YANG ; Jianyong LIAO
Chinese Journal of General Practitioners 2018;17(2):114-119
Objective To investigate the clinical features of patients with bronchiectasis of different types.Methods One hundred and twenty two patients with bronchiectasis at stable stage were recruited from January 2014 to July 2015.The patients were typed as cystic bronchiectasis (n =45) or non-cystic bronchiectasis (n =77) by high resolution CT (HRCT),expectoration bronchiectasis (n =80) or dry brochiectasis (n =42) by clinical symptoms,bacterial colonization (n =42) or non-bacterial colonization (n =80) by sputum culture.The modified British Medical Research Council (mMRC) dyspnea scale,Leicester Cough Questionnaire (LCQ),St George's Respiratory Questionnaire (SGRQ) and pulmonary function test were used to assess the clinical features,and the episodes of exacerbations and hospitalization,and mortality during 1-year follow-up were documented.Results mMRC dyspnea scale (1.90 ± 0.94 vs.2.90±1.09,t=-5.040),LCQ (16.20±4.60 vs.11.20±2.20,t=8.114),SGRQ (36.80±13.10 vs.52.06±22.10,t=-4.780),FEV1% pred (68.45 ±26.50 vs.52.22 ±20.60,t=3.458),FVC% pred (72.20 ±26.32 vs.63.10 ±21.42,t =2.058),FEV1/FVC (75.14 ±20.52 vs.58.12 ± 19.82,t =4.546),diffusing capacity of the lung for carbon monoxide (DLCO) (76.24 ± 28.40 vs.54.32 ± 21.20,t =4.400),episodes of exacerbations (Z =-8.272) and hospitalization during 1-year follow-up [6(14.29%) vs.29(36.25%),x2 =6.495] in patients with dry bronchiectasis were significantly better than those in patients with expectoration bronchiectasis (all P < 0.05).mMRC dyspnea scale (3.20 ± 2.10vs.2.10±1.40,t=3.131),LCQ (10.12±2.63vs.16.22 ±3.22,t=11.365),SGRQ (54.80± 18.12 vs.34.06 ± 12.10,t =6.839) and FEV1% pred (46.52 ± 22.55 vs.58.22 ± 24.62,t=-2.611),FVC% pred (60.24± 18.22 vs.70.10±24.20,t =-2.547),FEV1/FVC (62.54± 19.02vs.73.12 ±18.42,t=-3.025),DLCO (62.24 ±22.40 vs.74.52 ±26.26,t=-2.627),episodes of exacerbations (Z =10.213) and hospitalizations during 1-year follow-up [21 (46.67 %) vs.14 (18.18%),x2 =1 1.260] in patients with cystic bronchiectasis were significantly more severe than those in patients with non-cystic bronchiectasis (all P < 0.05).mMRC dyspnea scale (2.38 ± 1.45 vs.1.92 ± 1.14,t =2.175),LCQ (12.82 ±2.12 vs.16.20 ±3.96,t =-6.140),SGRQ (54.22±21.50 vs.41.20 ± 14.60,t =3.521) and FEV1 % pred (54.20 ± 21.60 vs.66.45 ± 28.24,t =-2.668),FVC% pred (63.10 ±24.32 vs.73.46 ±25.30,t =-2.177),FEV1/FVC (62.22 ±20.80 vs.72.14 ±24.36,t =-2.243),DLCO (58.52 ± 20.42 vs.69.22 ± 25.60,t =-2.344),episodes of exacerbation (Z =19.352) and hospitalization during 1-year follow-up [19 (45.24%) vs.16 (20.00%),x2 =8.575] in patients with bacterial colonization bronchiectasis were significantly more severe than those in patients with non-bacterial colonization bronchiectasis (all P < 0.05).However,there was no significant difference in mortality during 1-year follow-up (all P > 0.05) among patients with different types of bronchiectasis.Conclusion Patients with cystic,bacterial colonization and expectoration types of bronchiectasis seem to have more severe symptoms,more episodes of exacerbations and hospitalizations than those of non-cystic,non-bacteria colonization and dry types of bronchiectasis.
10.Construction of a lentiviral vector carrying short?hairpin RNA targeting PAX6 and its effect on proliferation of glioma U251 cells in vitro.
Xiao-Hong LIAO ; Wei-Lan YIN ; Fang WANG ; Li-Xiang WU ; Bai-Sheng HUANG
Journal of Southern Medical University 2017;37(12):1603-1608
OBJECTIVETo construct a lentiviral vector for delivering short hairpin RNA (shRNA) targeting PAX6 and investigate its effect on the proliferation of glioma U251 cells in vitro.
METHODSTwo small interfering RNA sequences targeting PAX6 gene were designed based on the reported sequence of PAX6 and annealed to form a double?stranded chain, which was inserted into a lentiviral vector to construct the recombinant lentiviral vector shRNA?PAX6. The recombinant vector was infected into U251 cells, and the expression of PAX6 mRNA and protein in the cells was detected by real?time PCR and Western blotting, respectively. The changes in the proliferation of U251 cells after the infection was assessed using MTT assay.
RESULTSDouble enzyme digestion of the lentiviral vector pLKD?CMV?G&NR?U6?shRNA yielded an 8208?bp fragment, and colony PCR and sequencing analysis confirmed successful construction of the lentiviral vector shRNA?PAX6. Infection of the cells with shRNA?PAX6 caused a significant reduction of the expressions of PAX6 mRNA and protein (P<0.05) and resulted in obviously increased proliferation of U251 cells (P<0.05).
CONCLUSIONWe successfully constructed the recombinant vector shRNA?PAX6 for silencing PAX6 gene. PAX6 gene silencing results in increased proliferation of U251 cells in vitro.

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