BACKGROUND: The glial scar formation after spinal cord injury in mammals is the physical and chemical barriers for neural regeneration, and relieving or delaying glial scar formation can provide benefit conditions for the regeneration of injured spinal cord.OBJECTIVE: To design and screen short hairpin RNA (shRNA) interfere molecular targeting the gene coding region of glial fibrillary acidic protein (GFAP) in rat, and reconstruct the eukaryotic vector of shRNA.DESIGN: An observational animal experiment.SETTING: Department of Spine Surgery, Shenzhen Hospital affiliated to Southern Medical University.MATERIALS: Twenty-five Wistar rats of clean degree, either male or female, weighing 20-25 g, were used. DMEM/F12,lipofectamine2000, Trizol RNA separating kits); fetal bovine serum (Hyclone); BamH Ⅰ, HindⅢ, Pstl, Sail and T4 ligases;Plasmid mini preparation kit and DNA gel extraction kit.METHODS: The experiments were carried out in the Shenzhen Hospital affiliated to Southern Medical University from October 2005 to June 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of GFAP target sequence with 9 bp spacer were designed and synthesized, then they were inserted directionally into plasmid Psilencer 2.1 respectively to generate small interfering RNA (siRNA) eukaryotic expression vector. ShRNA molecules were transfected by liposome via ex vivo expression repressive model of GFAP of rat spinal astrocytes. The effects of expressive suppression were detected with real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and then the optimal shRNA eukaryotic vector of repressive expression of GFAP was screened.MAIN OUTCOME MEASURES: ① Interfering sequence specific shRNA template synthesis; ② Constructing specific recombinant plasmid eukaryotic expression vector. ③ Culturing rat spinal astrocytes in vitro; ④ Effects of expressive suppression on GFAP in primary astrocytes after RNA interference detected with real-time fluorescence quantitative RT-PCR and Western blot.RESULTS: Sequence analysis showed that GFAP-shRNA recombinant plasmid eukaryotic expression vector was successfully constructed, and optimal GFAP-shRNA eukaryotic vector was screened using real-time fluorescence quantitative RT-PCR and Western blot. The GFAP expressions were reduced by 81%, 63% and 56% at the levels of mRNA and protein respectively.CONCLUSION: GFAP-shRNA eukaryotic expression vectors were successfully constructed and screened. The gene expression GFAP of primitive rat astrocyte can be suppressed significantly by the GFAP-shRNA eukaryotic expression recombinant optimized via ex vivo cellular expression suppression model, which should pave the way for the following multiple targets of RNAi genetic manipulation in the treatment of suppression of glial scar formation after spinal cord injury.

BACKGROUND: It is demonstrated that bone marrow stem cells (BMSCs) can generate neurosphere structures, which is similar to cloning sphere of neuron-specific enolase (NSE), in a specially induced system in vitro; therefore, BMSCs draw more and more attention as seed cells to repair central nerve injury.OBJECTIVE: To investigate the differentiation from BMSCs into neuron-like calls induced by fetal spinal cord tissue.DESIGN: Observational study.SETTING: Department of Spine Surgery, Second People's Hospital of Shenzhen.MATERIALS: SD rats (16 pregnant days old and 2 months old) were provided by Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Single antibody of NSE, multi-antibody of glial fibrillary acidic protein (GFAP) and single antibody of neurofilament (NF200) were provided by Wuhan Boster Company.METHODS: The experiment was carried out in Immune Opening Laboratory and Central Laboratory, Basic Medical Department, Tongji Medical College, Huazhong University of Science and Technology in September 2006. Bones of lower extremities of rats were collected to centrifuge BMSCs. Fetal spinal cord tissue homogenate was extracted from 16 pregnant days old rats to make inducing solution and induce differentiation of BMSCs. Otherwise, embryo muscle tissue was used to make muscle tissue homogenate as the same way so as to regard as the controls.MAIN OUTCOME MEASURES: BMSCs underwent morphological observation after induction; in addition, anti-NSE, NF200 and anti-GFAP were used to label neurons and astrocytes, respectively. Ten non-overlapping sights were randomly selected from positive reactive-induced cells after immunohistochemical staining under optic microscope to calculate ratio of positive cells of NSE and NF200 counting for total numbers of cells.RESULTS: ① Morphological changes of BMSCs after induction: During early induction, optic microscope indicated that soma of partial cells rebounded; whose apophysis was long and thin; apophysis of differentiated cells grew gradually and intercrossed each other. It was similar to nerve cells and some branches were similar to dendrite branches. However,morphological changes of cells in the control group were not obvious. ② Expression of relevant antigens differentiated from BMSCs into neuron-like cells at one week after induction: Most cells in spinal cord homogenate group expressed as positive NSE and NF200; a few of cells expressed as GFAP. While, positive staining of nerve cell antibody was not observed in the control group; meanwhile, positive reaction of nerve cell antigen was not observed in the control group,too. Immunohistochemistry examination demonstrated that positive rates of NSE and NF200 expressions were (68±1.7)%and (76.2±2.9)%, respectively.CONCLUSION: Fetal spinal cord tissue homogenate can induce differentiation from BMSCs into neuron-like cells.

Objective To evaluate the diagnosis and surgical treatment of primary hyperparathyroidism (PHPT). Methods 53 patients with PHPT who were treated in our hospital from 1997 to 2009 were analyzed retrospectively. Results All patients showed hypercalcemia and elevated parathyroid hormone (PTH). Among them, 43 were diagnosed as parathyroid adenoma, 6 were parathyroid hyperplasia and 4 were parathyroid cancer.The accuracy rate of parathyroid tumor localization was above 94. 3%. All patients presented temporary hypocalcemia after surgery. Conclusions Parathyroidectomy is an effective approach for patients with PHPT. Preoperative localization is essential to the surgery.

Objective To explore operation method,skills,and curative effect of orthophofia microscope discectomy(OMD)for the treatment of lumber disc herniation.Method Retrospective analysis of the 64 patients(74 herniated discs)of lumbar disc herniation heated by OMD,which included 38 segments posterior-lateral extrusion,32 segments central type herniated,and 4 segments extra-lateral extrusion.Resuits All patients were followed-up 6 to 8 months,average 7 months,54 cases were good and 4 cases were fair and no case was bad according to the Nakai scale.The choiceness rate was 93.75%,and no serious complications.Conclusions Orthophoria microscope discectomy has a better third dimension,good operation field of vision,less invasion and best clinical curative effect.Orthophoria microscope discectomy may be the best way of discectomy for the treatment of lumber disc hemiation.

Objective: To investigate the role of indoleamine 2, 3-dioxygenase in the development of uterine cervical squamous carcinoma. Methods: From January 2008 to December 2008, 116 uterine cervical carcinoma specimens and 18 metastatic lymph node specimens from patients with CIN Ⅰ-Ⅲ and uterine cervical squamous carcinoma were evaluated for iDO expression by immunohistochemistry. Twenty normal cervical specimens and 20 normal lymph node specimens were used as the controls. Results: The expression of IDO was not found in normal cervix and CIN Ⅰ. In CIN Ⅱ, IDO expres-sion was weakly positive in 2 cases (2/10, 20%) and negative in 8 cases (8/10, 80%). In CIN Ⅲ, IDO expression was weak-ly positive in 8 cases (8/13, 61.5%), positive in 1 case (1/13, 7.7%) and negative in 4 cases (4/13, 30.8%). The positive ex-pression rate of IDO in cervical cancer stage Ⅰ -Ⅳ was 100% (83/83). In cervical cancer stage Ⅰ A and Ⅰ B, the positive ex-pression rate of IDO was significantly higher than that in CIN Ⅱ and CIN Ⅲ (P<0.01). The positive expression rate of IDO in cervical cancer stage Ⅱ A-Ⅳ B was significantly higher than that in Ⅰ A and Ⅰ B. IDO expression was associated with cervi-cal cancer progression (OR=0.807, P<0.01). IDO expression in primary lesions with lymph node metastasis was significant-ly higher than that in those without lymph node metastasis. IDO expression rate was 100% in metastatic lymph nodes. The IDO expression was not associated with cervical squamous carcinoma differentiation degree (OR=-0.139,P>0.05). Conclu-sion: In CIN Ⅱ, escape mechanisms that stimulate cervical squamous carcinoma progression is gradually developed. IDO expression in metastatic lymph nodes is possibly associated with immune tolerance. IDO expression is not associated with differentiation degree of cervical squamous carcinoma. IDO may be a prognostic factor for uterine cervical squamous carci-noma and a therapeutic target for treatment.

BACKGROUND: Pathophysiological mechanism of local microenvironment is complex after central nerve injury; especially,both inflammatory reaction at an acute phase and formation of secondary glial scar have tremendous effects on effective regeneration of axon, regeneration and arrangement of local nerve cells, proliferation and migration of local stem cells;therefore, it becomes a basic reason for blocking nerve repair in an early period. Thus, how to effectively resist inflammatory factors in injured region at an acute phase and how to optimize microenvironment of neural regeneration are the most important strategies for repairing spinal cord injury in recent years.OBJECTIVE: To design, establish and screen the best expression of interleukin-6 receptor (IL-6R) α to inhibit shRNA adenovirus expression vector by using spinal cord injury models.DESIGN: Duplicative measurement study.SETTING: Department of Spine Surgery, the Second People's Hospital of Shenzhen.MATERIALS: A total of 40 healthy Wistar rats, either gender, 8-10 weeks old, were selected in this study. Rabbit-anti-rat glial fibrillary acidic protein (GFAP) antibody Ⅰ was provided by Santa Cruz Compan; siRNA eukaryon expression plasmid pGenesil (cohtaining green fluorescent expression system) was provided by Wuhan Jingsai Bioengineering Company.METHODS: The experiment was carried out in ImmuneOpening Laboratory, Basic Medical Faulty, Tongji Medical College, Huazhong University of Science and Technology, and Medica Laboratory Center, the Second People's Hospital of Shenzhen in November 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of IL-6 receptor (IL-6R) α target sequence with 9 bp spacer were designed and synthesized, then the recombinant adenovirus expression vectors with green fluorescence protein were constructed in vitro respectively. The acute spinal cord injury models were completed, and the adenovirus recombinants were regionally injected post 12 hours after spinal cord injury;in addition, the inhibitory effect of RNA interference (RNAi) on expression of IL-6R in local region after spinal cord injury were detected by using real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot so as to screen adenovirus expression vector which had the best inhibitory effect on expression of IL-6R.MAIN OUTCOME MEASURES: Inhibitory effect of RNAi on expressions of IL-6R RNA and protein in local region after spinal cord injury.RESULTS: Sequence analysis showed that IL-6R-shRNA recombinant adenovirus expression vector was successfully constructed, and optimal IL-6R-shRNA recombinant adenovirus expression vector was screened by using real-time fluorescence quantitative RT-PCR and Western blot. The IL-6R expressions were 49% and 56% at the levels of mRNA and protein, respectively.CONCLUSION: The IL-6R--shRNA recombinant adenovirus expression vector is successfully constructed and screened.The gene expression of IL-6R can be highly inhibited after acute spinal cord injury.

BACKGROUND: Glial cell-derived neurotrophic factor (GDNF) and transforming growth factor-beta 1 (TGF-β1)co-subordinate to TGF-β family. Both of them play very important roles in the development and differentiation of central and peripheral nervous system, and regulation of cell cycle in mammals.OBJECTIVE: To observe the differentiation of spinal cord-derived neural stem cells(NSCs) induced by GDNF combined with TGF-β1, and make a comparison of differentiation results with GDNF or TGF-β1 culture fluid.DESIGN: Controlled observation.SETTING: Central Laboratory, Shenzhen Hospital Affiliated to Southern Medical University.MATERIALS: Ten SD rats of clean grade, which were at conception for 16 days, were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technplogy. Main reagents and materials:DMEM/F12,B27(GIBCO); basic fibroblast growth factor (bFGF), GDNF; TGF-β1(PeproTech);fetal bovine serum (FBS,Hyclone); nestin multiple antibody (Boster, Wuhan); glial fibrillary acidic protein (GFAP) multiple antibody; neurofilament protein (NF-200) monoclonal antibody (Sigma).METHODS: This experiment was carried out in the Central Laboratory, Shenzhen Hospital Affiliated to Southern Medcial University between October 2005 and September 2006. Under the aseptic condition, rat fetus was isolated for isolation and culture of spinal cord-derived neural stem cells. In this study, five groups were divided: basal medium group, control group, bFGF group, TGF-β1 group, GDNF+ TGF-β1 group. In the basal medium group, DMEM/F12 containing penicillin,streptomycin, amphotericin (AMPH) B and 0.02 volume fraction of B27 annex solution. At 1 week after primary culture, rat spinal cord-derived NSC clones proliferated in vitro stably were harvested. In the control group, 0.1 volume fraction of FBS was added into basal medium. In the later three groups, induced medium was exchanged, i.e. 20 μg/L bFGF, 2 μg/L TGF-β1, and 10 μg/L GDNF+2 μg/L TGF-β1 were added into the basal medium, respectively. ①The differentiation of spinal cord-derived NSCs induced by different factors were observed under the optical microscope. ②The expressions of neurons and astrocytes were detected by immunocytochemical staining labeling. ③ The differentiated cells were counted by sorting technique by means of fluorescence excitation flow cytometer, and the percentage of NSCs differentiating into neurons and astrocytes were detected under the different induction environments.MAIN OUTCOME MEASURES: ① Morphological feature of cell differentiation in each group. ② Immunohistochemical detection of NSCs in each group. ③ The percentage of NSCs differentiating into neurons and astrocytes in each group.RESULTS: ① Cell morphology during differentiation: At the early stage of differentiation, lots of cells creeped to all the directions, and one week later, most of the migrated cells adhered to the wall entirely. Neuron-like cells, astrocyte-like cells and oligodendrocyte-like cells could be identified in the low-density cell region. ②Immunohistochemical detection results: A lot of GFAP- positive astrocytes were found in the control group and TGF-β1 group; Many differentiated neurons and NF-200 staining positive were found in the bFGF group and GDNF+ TGF-β1 group. ③Percentage of stained neuron and astrocyte: at one week of induction, the percentage of stained neurons was higher in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.15,19.56,25.32,P ＜ 0.05-0.01), and the percentage of stained astrocytes was lower in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.45,23.79,P ＜ 0.01 ).CONCLUSION: The combined in vitro induction of GDNF and TGF-β1 contributes to the neuronal differentiation of spinal cord-derived NSCs, indicating that both of them have synergistic effect.

Adult ; Aged ; Aged, 80 and over ; Bone Nails ; Bone Screws ; Equipment Design ; Female ; Femoral Neck Fractures ; diagnostic imaging ; surgery ; Follow-Up Studies ; Fracture Fixation, Internal ; instrumentation ; methods ; Humans ; Injury Severity Score ; Male ; Middle Aged ; Probability ; Radiography ; Retrospective Studies ; Treatment Outcome ; Wound Healing ; physiology

Objective To evaluate the optimum compatibility of nalbufine combined with ropivacaine for patient-controlled epidural analgesia (PCEA) after cesarean section.Methods A total of 100 parturients who were at full term with a singleton fetus,aged 24-35 yr,with body mass index of 29-33 kg/m2,of American society of Anesthesiologists physical status Ⅱ,scheduled for elective cesarean section under combined spinal-epidural anesthesia,were divided into 4 groups (n =25 each) using a random number table method:sufentanil 0.5 μg/ml plus 0.15％ ropivacaine group (SR group),nalbufine at final concentration of 0.2 mg/ml plus 0.15％ ropivacaine group (N1 R group),nalbufine at final concentration of 0.4 mg/ml plus 0.15％ ropivacaine group (N2R group) and nalbufine at final concentration of 0.4 mg/ml plus 0.1％ ropivacaine group (N3Rgroup).PCEA solution was prepared correspondingly after surgery,and all the drugs were diluted to 100 ml in normal saline in each group.The PCA pump was set up to deliver a 0.5 ml bolus dose with a 15-min lockout interval and background infusion at 2 ml/h.Visual analog scale scores of incisional pain and anduterine contraction pain were maintained＜4.Ramsay sedation scores were recorded at 8,12,24 and 48 h after surgery.The total pressing times of PCEA were recorded within 48 h after surgery.The development of adverse reactions such as nausea,vomiting,skin itching,numbness of lower extremity,urinary retention and respiratory depression was recorded in the analgesia period.Venous blood samples were collected before surgery and at 24 and 48 h after surgery for determination of plasma prolactin concentrations,and the time of colostrum was recorded.Neonatal nerve and adaptive capacity was assessed and scored.Results Compared with group SR,the total pressing times of PCEA were significantly reduced in N2R and N3R groups (P＜0.05),and no significant change was found in the total pressing times of PCEA in group N1R (P＞0.05).Compared with group N1R,the total pressing times of PCEA were significantly reduced in N2R and N3 R groups (P＜0.05).There was no significant difference in the total pressing times of PCEA between group N2R and group N3R (P＞0.05).The Ramsay sedation score was 2 in four groups.There was no significant difference in numbness of lower extremity,plasma prolactin concentrations or neonatal nerve and adaptive capacity scores among four groups (P＞0.05).Conclusion Nalbufine at final concentraction of 0.4 mg/ml mixed with 0.1％ ropivacaine is the optimum compatibility when used for PCEA after cesarean section.

Background: Post - stroke constipation is a commonly seen complication, which can reduce the quality of life of patients. Therefore, it is particularly important to explore the treatment of post-stroke constipation. Aims: To explore the effect of gastrointestinal rehabilitation (visceral fascial manipulation plus middle - frequency electrical stimulation) on quality of life, substance P (SP) and 5-hydroxytryptamine (5-HT) in constipation patients after stroke. Methods: Seventy constipation patients after stroke from Oct. 2020 to Oct. 2011 at Shanghai Tongren Hospital were enrolled and randomly divided into control group (lactulose treatment) and treatment group (gastrointestinal rehabilitation treatment). Montreal cognitive assessment (MoCA), Barthel index (BI), self-rating anxiety/depression scale (SAS/SDS), gastrointestinal symptom rating scale (GSRS), Wexner constipation scale, and patient assessment of constipation-quality of life (PAC-QOL) were performed before and after treatment. Serum SP, 5 - HT were determined by ELISA. Results: After the treatment, no significant difference in MoCA score was found in both two groups, and SAS score, SDS score, GSRS score, Wexner score and PAC-QOL score were significantly decreased (P<0.05). Repeated measures ANOVA showed that significant differences in SAS score, SDS score were found between the two groups (P<0.05), and the decreases of SAS score and SDS score were more obvious in treatment group. No significant differences in MoCA score, BI score, GSRS score, Wexner score and PAC- QOL score were found between the two groups (P>0.05). Serum SP, 5-HT levels in treatment group were significantly higher than those in control group (P<0.05). Conclusions: In the treatment of constipation after stroke, the gastrointestinal rehabilitation therapy of‘visceral fascial manipulation plus middle-frequency electrical stimulation’has the therapeutic effects equivalent to drug therapy, and is worthy for promoting in clinical treatment.