Objective To study the effects of muscular trumatic fluid on the biological properties of muscle-derived stem cells (MDSCs) in rats.Methods MDSCs were isolated and purified by the preplate technique,muscle injury was made for the extraction of muscular traumatic fluid.Bradford colorimetric was used to detect the protein content in the muscular traumatic fluid,and the fluid with the highest protein was used to co-culture with MDSCs.Methyl thiazolyl tetrazolium (MTT) assay and wound model of monolayer of cells were used to observe the effects of wound fluids on the proliferation and mobility of MDSCs.The expressions of α-SMA and Vimentin were tested by immunohistochemistry and Western blot technique.Results MTT assay showed that wound fluid with concentration of 10% could most significantly increase the proliferation of MDSCs.The increased expressions of α-SMA and Vimentin were found in MDSCs after cultrue with muscular traumatic fluid in vitro,and the time-dependent relation exists.Conclusions Wound environment can directly participate in the muscle regeneration by inducing the proliferation,mobility of MDSCs;on the other hand,MDSCs can differentiate into fibrotic cells under stimulation of muscular traumatic fluid.

OBJECTIVE: To establish an HPLC method for the determination of plasma concentration of Irbesartan tablets in rats and its pharmacokinetics study.METHODS: The determination was performed on Cosmosil-C18 column with mobile phase consisted of phosphoric acid-methanol-acetonitrile(7 ∶ 80 ∶ 13,V/V/V) at flow rate of 1.0 mL?min-1.The detection wavelength was set at 272 nm.Rats were given Irbesartan solution 30 mg?kg-1 via i.g.gtt.The plasma concentration of irbesartan were determined before medication and at 0 min,5 min,10 min,20 min,30 min,45 min,60 min,120 min,180 min,240 min,360 min,480 min after medication.The pharmacokinetics parameters were estimated.RESULTS: The linear range of irbesartan was 0.5～50 ?g?mL-1(r=0.999 1) with an average recovery of 93.89%(RSD

Objective To construct a lentiviral vector expressing small-hairpin RNA(shRNA) targeting Smad3 gene.Methods The targeting sequence of Smad3 gene which can be effectively silenced in RNA inference was confirmed in our previous study.The cDNA containing both sense and antisense Oligo DNA fragments of the targeting sequence was designed,synthesized and cloned into the pGCSIL-GFP vector.The obtained lentiviral vector containing Smad3 shRNA was confirmed by PCR and sequencing.293T cells were cotransfected with lentiviral vector GC-shSmad3,pHelper 1.0 and pHelper 2.0.The titer of virus was tested according to the expression level of GFP.Results PCR and DNA sequencing demonstrated that the constructed lentivirus vector GC-shSmad3 produced Smad3 shRNA.The titer of concentrated virus was 3?108 TU/ml.Conclusion The lentivirus RNAi vector targeting Smad3 is constructed successfully.

Objective To establish a co-culture system between mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs) in vitro, and study the influence of SMCs on the differentiation of bone marrow MSCs (BMSCs) into SMCs in the co-culture system. Methods Density gradient centrifugation was used to separate and culture BMSCs, and collagenase digestion was employed to separate and culture bladder smooth muscle cells(BSMCs). Then the 2 types of cells were identified respectively. Then the cells were divided into 4 groups, BMSCs culture group (positive control), BSMCs cells culture group (negative control), BMSCs and BSMCs co-culture groups with contact or non-contact. The expression of ?-smooth muscle actin (?-SMA) and calponin were detected by immunofluorescence staining. Western blot analysis was employed to determine protein expression of calponin. Results In contact co-culture group, the positive rate of CD90 abd CD44 were 99.78% and 60.27% respectively in BMSCs, while only 0.21% for CD45. Western blotting showed that BMSCs expressed little calponin, but those contacting with BSMCs had an increasingly stronger expression of calponin with the elapse of contact culture time, while there was no significant change in its expression in BMSCs with contact. Immunofluorescence staining indicated that part of BMSCs were calponin positive at the 8th day after culture, but no positive cell was observed in non-contact culture group and BMSCs culture group. Conclusion The contact between BSMCs and BMSCs can promote the differentiation of BMSCs into SMCs.

Objective To study the desired therapeutic effect of vaginoplasty using sigmoid colon or peritoneum for entire vaginal atre-sia.Methods From May 2015 to October 2015,8 patients with complete vaginal atresia in our hospital performed vaginoplasty using sigmoid colon or peritoneum successfully.The operation methods,prognosis and therapeutic effect and quality of sexual life of two groups were com-pared and analyzed to investigate a perfect operative method and ideal nursing.Results Laparoscopic vaginoplasty using the peritoneum compared with that using sigmoid colon has advantages of shorter time of operation,less bleeding,less trauma and quicker recovery.However, artificial vagina using sigmoid colon was much more similar to a natural vagina in morphological and physiological characteristics,merely ac-companied by excessive mucus discharge during the postoperative period.At lower risk of vaginal adhesion and stenosis,laparoscopically as-sisted vaginoplasty using sigmoid colon graft also puts high technical requirements.Two patients normally developed uterus menstruate regular-ly.Four patients without uterus and 2 patients with rudimentary uterus experienced good sexual intercourse after vaginoplasty.Conclusion Patients with complete colpatresia who have normal womb should creat a patent and functional vagina until adolescence to restore its physio-logical and reproductive function.Anatomical reconstructive treatment of vaginal agenesis for patients with absent or hypoplastic uterus can be postponed till the late teens or in the adult.Both laparoscopic sigmoid vaginoplasty and peritoneal vaginoplasty achieve a minimal-invasive, cosmetic,natural lubricous, smooth artificial vagina for patient’ s sex life satisfactory.

A method was developed for the determination of tetrodotoxin in marine organisms by high perfor-mance liquid chromatography-mass spectrometry with immunoaffinity column. The samples were extracted with 1% acetic acid methanol solution and diluted with phosphate buffer at pH 7-8. After cleaned up by immuno-affinity column, the samples were analyzed by LC-MS/MS and quantitatively determined by external standard method. The chromatographic separation was performed on an ACQUITY UPLC BEH Amide column with gradient elution by using acetonitrile and 5 mol/L ammonium acetate solution containing 0. 1% formic acid as mobile phase. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode. Linear ranges of TTX was in the range of 0. 3 -20. 0 μg/L with correlation coeffi-cient more than 0. 997. The quantification limit of the method was 0. 3 μg/kg. The recoveries of standard addition for tetrodotoxin were 88. 7%-102. 3%, and the relative standard deviation was 2. 0%-6. 4%. The method could be used to identify and quantify tetrodotoxin in marine organisms with satisfactory reproducibility and sensitivity.

Objective:To explore the advantages and value for clinical application of clavicle segment osteotomy approach with pectoralis major muscle pedicle in surgical resection of posterior brachial plexus tumour.Methods:From April 2010 to December 2020, 6 patients with brachial plexus tumour behind the clavicle were treated. Two patients had the tumour on the left brachial plexus and 4 on the right. Two patients had the tumours located in the upper trunk of brachial plexus, 2 in the middle trunk, 1 in the medial plexus and 1 in the posterior bundle. The neurological function evaluation were grade II in 3 cases and grade III in 1 case preoperatively. The tumours sized from 3.0 cm× 3.0 cm×2.0 cm to 11.0 cm×8.0 cm×6.0 cm. The clavicular segment osteotomy approach with the pedicle of the pectoralis major was applied in order to expose the surgical field and remove the nerve tumour. All patients underwent regular postoperative outpatient clinic follow-up to record the sensation of the affected limb and the recovery of muscle strength, tumour recurrence and fracture healing.Results:In the operation, it was found that there was still a little glial-like tissue in the nerve sheath after complete dissection of the tumours in 2 patients. Postoperative pathological examination reported that there were 4 schwannoma, 1 malignant peripheral schwannoma and 1 neurolipoma. The postoperative follow-up lasted for 6 to 34 months, with an average of 12.8 months. All patients showed no symptoms of brachial plexus injury or tumour recurrence. The clinical symptoms were relieved or disappeared. The neurological function evaluation reached grade III in 5 patients and grade IV in 1 case. At the same time, the internal fixation of the clavicle was satisfactory and achieved bone union in all cases. Fracture healing time was 2.5 to 4.5 months, with an average of 3.2 months. The internal fixation was removed from 3 patients after operation in 1 year. No obvious limb movement disorder and periarthritis of shoulder and other complications occurred.Conclusion:The transclavicular osteotomy approach with the pedicle of the pectoralis major can fully expose the anatomical alignment of the brachial plexus and the relationship adjacent and between the tumour and the surrounding tissues in the surgery for a brachial plexus tumour behind the clavicle, which is helpful for a complete tumour resection. It is a feasible method to treat brachial plexus tumour behind the clavicle by finding residual tumour-like tissue, reducing the risk of surgery and tumour recurrence, and providing excellent blood supply to the osteotomy segment of the clavicle to promote fracture healing.

Objective:To investigate feasibility and clinical effect of free anterolateral thigh flap(ALTF) with reversed arterial flow in repair of foot and ankle wounds in children.Methods:From October 2014 to February 2021, the free ALTF with reversed arterial flow was used to repair the wounds in 7 children with severe soft tissue injury and main blood vessel injury in the Department of Hand & Foot Surgery of the Second Affiliated Hospital of University of South China. Of the 7 injured children, 5 were caused by traffic accident, 1 by mechanical strangulation and 1 by heavy object. The size of wounds ranged from 6.0 cm×8.0 cm to 9.0 cm×11.0 cm. ALTF were designed to be anastomosed with the blood vessels at the recipient sites by retrograde blood supply. The size of flaps ranged from 6.0 cm×8.0 cm to 10.0 cm×11.0 cm. All flap donor sites were pulled together and directly sutured. Follow-ups were conducted through outpatient clinic visits, telephone call and WeChat reviews in the 1st, 3rd, 6th, 12th and 24th months after surgery. The major contents in follow-up were the shape, colour, texture, sensation of flap and function of ankles.Results:All donor sites healed at I stage. A total of 5 flaps survived successfully; One flap had venous occlusion after surgery, and the flap survived after surgical exploration; One flap had partial necrosis after surgery, and repaired with artificial dermis after further debridement. Postoperative follow-up lasted for 6-24 months, with an average of 10.5 months. All flaps appeared in similar colour and texture to the surrounding soft tissues. Two children underwent flap repair after half a year due to bloated flaps. The Ankle-hind foot Function Score of American Orthopedic Foot Ankle Society(AOFAS) was used to evaluate the ankle function. AOFAS achieved 84-94 points, with 5 children in excellent and 2 in good. The sensation recovered to S 3+ in 5 children, S 3 in 1 child and S 2 in 1 child, according to the standard of British Medical Research Council (BMRC). Conclusion:For the children with severe soft tissue injury combined with main vascular injury in foot and ankle, free ALTF with reversed arterial flow can repair the defect and effectively secure the blood supply. It is a feasible method for wound repair.