Objective To observe the immunoregulatory roles of MSCs in inducing proliferation of B regulatory cells (Bregs).Method DA Rat MSCs were co-cultured with Wistar rat B cells.The levels of IL-10 and secrete proinflammatory cytokines in the supematants were determined by using ELISA.The proliferation and apoptosis of B cells after interaction with MSCs were analyzed by using FACS.In order to investigate whether Bregs are involved in the phagocytosis of immune complex (IC),B cells and Bregs were incubated with FITC-OVA-IC.The fluorescence intensity was detected by using immunofluorescence microscopy.Results MSCs induced B cells to differentiate into Bregs with high CD19,CD1d and CD5 expression and high IL-10 level.The proliferation of B cells was significantly suppressed after interaction with MSCs.Bregs had enhanced phagocytic capacity as compared with that of B cells.Bregs significantly inhibited maDC-induced CD4+ T cell proliferation compared to B cells,without depressing IL-2 or IFN-γ secretion.Conclusion MSCs induce freshly isolated B cells to differentiate into Bregs characterized by the ability to preferentially produce IL-10.

Objective To construct lentiviral vector miR30-CD133 targeting CD133 gene and investigate its effects on SMMC7721 cells. Methods Thespecific sequence of DNA which containing both sense and antisense Oligo DNA of the targeting sequence were designed, synthesized and cloned into the pPRIME vector. pPRIME-miR30-CD133, psPAX2 and pMD2G were co-transfected on 293T cells to get the lentivirus the transfection efficiency was investigated by confocal laser scanning microscope. The expression of miR30-CD133 on CD133 were detected by qRT-PCR and Western blott. The proliferation and apoptosis effect of miR30-CD133 was respectively evaluated by CCK-8 assay and AnnexinV/PI. Results Functional PFU titers of PRIME-miR30-CD133 were 6.58 × 109 PFU/mL. The expression of CD133 mRNA in sh CD133 group (0.18 ± 0.04) was less than Blank group and shNC group (P<0.05). The expression of CD133 protein in sh CD133 group was less than Blank group (10%) and shNC group (8%) (P < 0.05). Cells proliferation activity were significantly inhibited in shCD133 group compared with control group. Apoptosis rate were significantly increased in shCD133 group (41.3%) compared with Blank group (25.3%)and shNC group (24.3%). Conclusion The lentivirus miR30 vector targeting CD133 gene was successfully constructed, which will be a basis to the further CD133 gene studies.

Objective To study the surgical techniques,indications and clinical effects of microendoscopic lumbar discectomy with the preservation of the ligamentum flavum.Methods A total of 65 patients with lumbar disc herniation were enclosed in the study.All the patients suffered from low back pain and radicular syndrome.The diagnosis was confirmed with CT scanning and/or MRI examination.The Microscope Endoscopic Tubular Retractor System(METRx) was used to access the interlaminar space.The superior,inferior and lateral edges of the ligamentum flavum were released using a micro-scalpel.The nerve root was retracted medially with a nerve root retractor to expose the herniated lumbar disc for performing the discectomy.The dissociative ligament was restored anatomically after disc removal and the decompression of the nerve root.Results The operation was completed smoothly in all the 65 patients without conversions to open surgery.The operation time was 136?21 min(range,110~170 min).The wound healed by first intention in all the patients.No nerve root injuries,intervertebral infection,or cerebrospinal fluid leakage were observed.A follow-up was carried out for 6~24 months(mean,14.5 months).According to the Nakai classification,excellent results were achieved in 42 patients,good in 18 patients,fair in 3,and poor in 2,the rate of excellent or good outcomes being 92.3%(60/65).Conclusions Microendoscopic lumbar discectomy with the preservation of the ligamentum flavum using the METRx is feasible.The preserved ligamentum flavum,as a good natural barrier,is helpful to prevent epidural fibrosis.

[Objective]To explore the feasibility and efficiency of treatment of lumbar degenerative disease with unilateral decompression,interbody fusion and percutaneous pedicle screw fixation under endoscopic system.[Method]From June 2004 to March 2007,20 patients underwent minimally invasive transforaminal lumbar interbody fusion(TLIF) or posterior lumbar interbody interbody fusion(PLIF),which consisted of 11 male and 9 female patients.The mean age was 46.2 years(range,31～70),and the preoperative diagnosis consisted of postoperative recurrent lumbar disc herniation(n =8),far lateral lumbar disc herniation(n =4),spinal stenosis(n = 3),lumbar instability(n = 3),and discogenic lumbar pain(n =2).One-level decompression and intebody fusion with unilateral pedicle screw fixation under endoscopic system was performed in all of cases(12 at L4、5,and 8 at L5S1).A paramedian,muscle-sparing approach was performed through a tubular retractor docked unilaterally on the facet joint.A total facetectomy was then conducted,exposing and removing the disc(TLIF),or microendoscopic discectomy(MED) was performed(PLIF).The intervertebral space preparation were completed through the X-tube or METRx system.Interbody fusion was achieved with autograft bone and interbody cages.Unilateral pedicle screw-rod placement was accomplished.[Result]There was no conversions to open surgery.Operative time averaged 115 minutes(range,100～165 min).Blood loss averaged 130 ml(range,50～180 ml).Mean length of postoperative hospital stay was 11 days(range,7～15 days).All patients presenting with preoperative low back pain and /or lower extremity radicular pain(n= 20) had resolution of symptoms postoperatively.Complications included two cases of new radiculopathy postoperatively(one from graft dislodgement,the other from hematoma formation).Twenty patients were followed up 10～39 months(average 21.6 months).The preoperative,1 month postoperative and last follow-up Oswestry Disability Index(ODI scores were 42.05+8.36,21.33?6.37 and 12.31?3.75 separately(P

[Objective]To explore the feasibility and safety of percutaneous kyphoplasty(PKP) and percutaneous vertebroplasty(PVP) treating osteolytic lesions of high thoracic vertebra with metastasis by one side extrapedicular approach,and to assess the clinical result of minimally invasive technique. [Methods]In March 2008,one patient(male,59 years old) with T1~3 vertebral metastases of lung cancer diagnozed 8 months ago was selected.The symptoms included extremely severe pain in upper thoracic spine and left should.The analgesic effect was limited for more than 6 months.There was no operative option.Domestic PKP and PVP tool systems were used in local anaesthesia.Under fluoroscopic guidance,T2 and T3 vertebral augmentation were separately completed by single side extrapedicular approach PKP and PVP.Clinical results were followed up and observed.[Results]The procedure was performed smoothly.T3 and T2 vertebrae were differently treated by PKP and PVP.T1 received no treatment because of patient`s intolerance.The operative time of T3 vertebral PKP was 57 minutes.The volume of injected bone cement was 1.9ml.The operative time of T2 vertebral PVP was 49 minutes.The volume of injected bone cement was 1.5ml.Extravertebral leakage of the polymethylmethacrylate(PMMA) into the paravertebral itssue was found without clinical symptom,because osteolysis occurred in the left pedicle of T2 vertebra.There was no other complication.The patient was discharged 5 days after operation.The preoperative,2 days and 3 months postoperative follow-up VAS scores were 10,3 and 6.The patient's markedly pain could be controlled by analgesia.[Conclusion]One side approach percutaneous kyphoplasty is a safe and effective technique for treatment of high thoracic vertebral metastasis with markedly relief of pain.

[Objective]To complete comparison between anterior cervical surgery by microendoscopic and open operation,explore feasibility and efficacy of anterior cervical decompression,interbody fusion and fixation by microendoscopic surgery,and give preliminary clinical evaluation of mieroendoscopic surgery.[Method]In a retrospective study,46 patients underwent one level cervical surgery by anterior approach.23 patients(23~64 years,41.5 years in average)were treated with microendoscopic surgery as microendoscopic group.Under general anesthesia,a transverse incision(1.6 cm)was made at right side of neck.A tubular retractor was then inserted and fixed,and a specially designed endoscope was placed inside the tubular retractor.Discectomy and interbody fusion with insertion of Cage or/and plate fixation was performed by endoscope.At fracture and dislocation patient group,titanium Cage was used in 1 case,CBK in 1 case,plate in 5 cases.At cervical disc herniation patient group,titanium Cage was used in 2 cases,CBK in 12 cases,plate in 2 cases.During the same period,23 patients(25~68 years,46.5 years in average)were treated with open surgery as open group.A transverse incision(4～5 cm)was made by right route approach.Discectomy and interbody fusion with Cage or/and plate fixation was performed by general procedure.[Result]At microendoscopic group,23 cases were followed up from 10 to 22 months(16.5 months in average),and mean operative time were 95 minutes,mean blood loss 90 ml.For fracture and dislocation patients,by Frankels classification,2 cases with complete tetraplegia showed no improvement,2 cases with incomplete tetraplegia improved from grade C to grade D postoperatively,1 case upgraded from C to E.For cervical spondylotic myelopathy patients with disc herniation,according to Odoms scoring system,10 cases had excellent outcome,5 good,1 fair.At open group,23 cases were followed up from 8~21 months(15.2 months as average),and mean operative time was 95 minutes,mean blood loss 90 ml.By Frankels classification,2 cases with complete tetraplegia had no improvement,3 cases with incomplete tetraplegia improved from C to D,1 case from D to E.According to Odoms scoring system,8 patients suffering from cervical spondylotic myelopathy with disc herniation had excellent result,6 good,2 fair.[Conclusion]Compared with open surgery,microendoscopic surgery with endoscopic instrument and technique can be used for one level discectomy,interbody fusion and internal fixation,and offer a similar short-term good clinical outcome with minimal incision,less traumatic reaction and postoperative discomfort.

Objective To isolate and culture the rabbit mesenchymal stem cells,and explore the biological features in vitro.Methods Two milliliter of bone marrow was extracted from the tibia of juvenile rabbit under anesthesia and sterile conditions,and mesenchymal stem cells were obtained by density gradient centrifugation and inoculated to 100ml culture flask.The morphological changes,adherence rate and ultrastructure of the stem cells were observed.The surface markers and growth cycle of the mesenchymal stem cells were detected by flow cytometry,and biological features were observed by inducing the cells differentiate into osteoblasts.Results The adherence of mesenchymal stem cells started 2 hours after culturing,basically finished 20 hours later,and more than 90% of the cells were fused at the 15th day.Observed with transmission electron microscopy,the cultured cells showed typical ultrastructure of stem cells: numbers of microvillus mainly distributed on one side of the cells,irregular nuclei with indentation,and lots of cell organs.Analysis for the cell surface markers indicated positive expression of CD44 and CD90 and negative expression of CD45 in mesenchymal stem cells.Cell growth cycle assay showed that most mesenchymal stem cells(about 97%) stayed in stage G1 and G2.Three weeks after osteoblastic induction,the mesenchymal stem cells gradually produced small calcium salt crystals,the cells were positive in alkaline phosphatase staining,and Von Kossa staining showed many calcium salts in the cell clone as black reaction deposits.Conclusions The rabbit bone marrow mesenchymal stem cells isolated by density gradient centrifugation are of high purity and stable growth rate,and the cells' characters can be still maintained after serial subcultivation.Since easy to operate on isolation and proliferation,the mesenchymal stem cells could be used as the tissue engineering seed cells in repairing of intervertebral disc degeneration.

Objective To summarize the perioperative complications of microendoscopic discectomy(MED)for lumbar disc herniation,and to discuss the therapeutic strategies for these complications.Methods From October 1999 to Decmeber 2006,1852 patients with lumbar disc herniation were treated by MED in our hospital,140 of them developed perioperative complications.The clinical data of these patients were analyzed.Results Hemorrhage from the vertebral venous plexus was found in 48 cases,among which,MED was completed after controlling the bleeding under an endoscope in 42 cases,open discectomy was carried out in the other 6.In 47 cases,the herniated disc was incorrectly localized,and the MED was completed after correcting the location and direction of the endoscope.Twenty-one cases developed rupture of the dura mater during the MED,2 of them were converted to open surgery.Thirteen patients had leakage of the nucleus pulpous,and received a second-stage MED to remove the spinal nucleus.Six patients developed nerve injury and recovered completely one month later.Intraspinal infection was found in 5 patients after the MED;one of them was cured by conservative therapy,and the other 4 recovered after receiving the evacuation of the intraspinal lesions.Conclusion Complications of MED for lumbar disc herniation can be prevented or reduced effectively by proper therapeutic strategies.

Objective To investigate the variation of serum Thymidine Kinase 1(TK1) in patients with non‐small cell lung canc‐er(NSCLC) and its clinical value .Methods Serum TK1 level of 129 patients with NSCLC and 90 healthy volunteers were measured by sensitive chemiluminescence dot‐blot assay .The serum TK1 level variation of 76 patients with NSCLC were compared before and after operation .Results Serum TK1 level of NSCLC patients were significantly higher than the level of healthy control group(P<0 .05) .Serum TK1 level in Stage Ⅲ >Stage Ⅳ >Stage Ⅰ + Ⅱ >Stage Tis ,comparison between each other was of statistical differ‐ence(P<0 .05) .The serum TK1 level of NSCLC patients at 30th day after surgery was remarkably lower than which before surger‐y(P=0 .001 ,P<0 .01) .The serum TKl level was correlated with lymphatic metastasis(P<0 .01) ,but not with other factors such as pathology types ,age ,sex and smoking .The serum TK1 measurement was high sensitivity and specificity to the diagnosis of NSCLC .Conclusion The serum TK1 level detection has important clinical significance in diagnosis and evaluation of NSCLC pa‐tients .

BACKGROUND: The commonly used culture methods for primary intervertebral disc cells are type Ⅱcol agenase alone digestion method, and type Ⅱ col agenase combined trypin digestion method. However, the acquired cells are few. OBJECTIVE: To acquire nucleus pulposus cells from human degenerative intervertebral disc using a systemic and simplified method. METHODS: Nucleus pulposus cells were isolated and cultured. The morphology of nucleus pulposus cells was observed under inverted phase contrast microscope every day. Primary and subcultured cellsuspension was applied for the determination of cel viability using trypan blue staining. The cel growth was detected with MTT assay. The cel morphology was observed under laser confocal microscopy. RESULTS AND CONCLUSION: Nucleus pulposus cells were successful y isolated from from human degenerative intervertebral disc using the improved method, and cells were subcultured to passage 3. Primary cells were fusiform shaped, while cells at passage 1 and 2 were triangle or polygonal, which were similar to fibroblasts. When cells almost reached the confluence, the cells showed slabstone-like appearance. Trypan blue staining showed that, the primary cel viability was 99%, and passage 3 cells had 93%-95% viability. The proliferation of nucleus pulposus cells gradual y decreased as the generation increased. Compared with passage 1 cells, passage 2 and 3 cells at logarithmic phase trended to be smoother. The cel morphology observed under laser confocal microscopy was similar to the results under phase contrast microscope. The improved simple method can successful y acquire a variety of cells from human degenerative intervertebral disc, and these cells show fine biological properties.