Objective To investigate the effects of intrathecal (IT) NaV 1.8 antisense oligonucleotide on the chronic neuropathic pain induced by chronic constrictive injury (CCI) to the sciatic nerve.Methods Twenty-four male SD rats were randomly divided into 4 groups ( n = 6 each): group Ⅰ CCI + NS 5 ?l; group Ⅱ CCI + mismatch oligonucleotide 45 ?g; group ⅢCCI + antisense oligonucleotide 45 ?g and group Ⅳ CCI + antisense oligonucleotide 90 ?g. CCI was produced by placing 4 loose ligatures on the left sciatic nerve at 1 mm interval with 4-0 chromic catgut as described by Bennett. On the 5th day after CCI IT catheter was inserted at the level of lumbar spine and identified by free flow of CSF. On the 8th day after CCI normal saline or mismatch or antisense oligonucleotide was injected IT twice a day for 5 consecutive days. Threshold to noxious thermal and mechanical stimuli was measured before CCI (baseline) and on the 1st, 3rd, 5th, 7th, 9th, 11th and 13th day after CCI. On the 14th day after CCI the lumbar segment of spinal cord was removed for determination of NaV 1.8 sodium channel expression in L4-6 dorsal root ganglion (DRG) by semi-reverse transcriptase-PCR.Results The threshold to von Frey hair stimulation and noxious thermal stimuli on the operated side was significantly lowered after CCI. On the 11th and 13th day after CCI the threshold to mechanical and thermal stimuli were significantly higher in group Ⅲand Ⅳ than in group I and H . Conclusion IT NaV 1. 8 antisense oligonucleotide can reduce the neuropathic pain by down-regulating NaV 1.8 mRNA expression.

Objective To discuss the survivorship of rat mesenchymal stem cells (rMSCs) and the expression of human brain-derived neurotrophic factor (hBNDF) protein after transplantation of the hBDNF-modified rMSCs (hBDNF-rMSCs) to the adult rats with spinal cord injury (SCI) and discuss the effect of hBDNF-rMSCs on the apoptosis of rat neural cells. Methods A total of 240 adult male Sprague-Dawley rats were randomly divided into sham operation group,SCI group,hBDNF-rMSCs transplantation group and empty vector-rMSCs transplantation group,with60 rats in each group.The SCI model was established by using the modified Allen technique.At day 7 after modeling,an equal volume of hBDNFrMSCs suspension,empty vector-rMSCs suspension and phosphate buffered saline (PBS) were injected through the L4,5 subarachnoid space into the hBDNF-rMSCs group,empty vector-rMSCs group and SCI group,respectively.Then,the injured spinal cord tissues were obtained from each group at days 1,2,3,7 and 14 after transplantation to observe the viability and distribution of rMSCs with enhanced green fluorescent protein gene by fluorescent microscope,measure the expression of hBDNF protein by Western blot and detect the apoptosis of neural cells by TdT-mediated dUTP nick end labeling (TUNEL). Results Both hBDNF-rMSCs and empty vector-rMSCs groups showed green fluorescence expression of rMSCs.The hBDNF protein expression was observed in hBDNF-rMSCs group and changed with time,ie,the expression was detected at day 2 after transplantation,reached the highest level at day 7 and then decreased gradually.Among the hBDNF-rMSCs,empty vector-rMSCs and SCI groups,the number of TUNEL positive cells was the least in hBDNF-rMSCs group,followed by the empty vector-rMSCs group and the number was relatively more in SCI group at days 2,3,7 and 14 after transplantation,with significant differences among groups (P ＜ 0.05). Conclusions Transplantation of the hBDNF-modified rMSCs through subarachnoid approach are able to survive and assemble at the injured spinal cord area and express hBDNF protein.The hBDNF-modified rMSCs can inhibit the apoptosis of neural cells after SCI.

Objective To investigate the effect of FK506 on the expression of axon guidance cue slit-2 after spinal cord injury(SCI)in rats.Methods A total of 75 adult Sprague-Dawley rats were randomly divided into three groups,ie,sham operation group,SCI group and FK506 treatment group.The SCI model was made by using the modified Allen's technique.Then,the rats were sacrificed and the spinal cord was removed at different time points(at days 1,3,7,14 and 28)for detection of the expression of slit-2 by means of reverse transcription polymerase chain reaction(RT-PCR)and immunohistochemistry.Results The expression of slit-2 changed with time.The expression of slit-2 could be detected at day 1 after SCI,reached the highest at day 7 and then decreased gradually,with higher expression level in the injury group compared with treatment group(P ＜ 0.05).Conclusion Following spinal cord injury,administration of FK506 can up-regulate the expression of slit-2 and may exert important effect on the guidance of the axon regeneration.

Objective To observe the effect of FTY720 on the expressions of Bel-2 and Bax protein and explore the neuroproteetive effect of FTY720 on neuron after acute spinal cord injury in the rats.Methods A total of 75 SD rats were divided randomly into three groups,ie,the test group(treated with FTY720),the injury group(treated with normal saline)and the control group.The test and injury groups were impacted to create T10 spinal cord injury(SCI)by Allen's method.Both the mRNA and protein expressions of Bcl-2 and Bax in the injured spinal cord section were studied respectively with hematoxylin and eosin(HE)staining,immunohistochemical examination and reverse transcription polymerase chain reaction(RT-PCR)technique at different time points(at6 h,12 h,24 h,3 d and 7 d). Results The expressions of Bcl-2 and Bax in the test group and the injury group were higher than that in the control group at all time points.Meanwhile,at 12 h,24 h,3 d and 7 d,the mRNA and protein expressions of Bcl-2 in the test group were significantly higher than that in the injury group(P<0.05),while the mRNA and protein expressions of Bax in the test group were obviously lower than that in the injury group (P<0.05). Condusion Early administration of FTY720(0.5 mg/kg)after spinal cord injmy Canincrease the mRNA and protein expressions of Bcl-2,decrease the expression of Bax and inhibit neuron apoptosis in the injured spinal cord.

Objective To evaluate the efficacy of the parathyroidectomy (PTX) in the treatment of severe secondary hyperparathyroidism (SHPT) with Sagliker syndrome (SS). Methods A retrospective review was undertaken among 212 SS patients underwent PTX in our hospital and with more than 3 years' follow up. The definitions of the efficacy were based on the postoperative intact parathyroid hormone level (iPTH). Cure showed that the iPTH was ＜ 150 ng/L; marked effectiveness was 150-300 ng/L; effectiveness was 301-500 ng/L;ineffectiveness was ＞500 ng/L. The status was defined as persistent SHPT if iPTH was ＞ 150 ng/L after surgery. The status was considered as SHPT recurrence if iPTH was ＜ 100 ng/L in the first week after surgery, and gradually increased and ＞ 150 ng/L with the follow-up. Results ( 1) Ten patients were involved and the average dialysis time was 142 months [male/female: 4/6; age 30-54 (39. 3 ± 10. 4) years]. All patients had severe bone and joint pain, accompanied with progressive facial increases, chicken breast, kyphosis, hip bone deformities, and body height shortening. (2) Preoperative tests: the median of iPTH 2000(1800-2863) ng/L; serum calcium (2. 45 ±0. 21) mmol/L, phosphorus (2. 19 ±0. 51) mmol/L, alkaline phosphatase ( ALP) (1189. 8 ± 780. 0) IU/L. Two to four enlarged parathyroid glands were confirmed by ultrasound and 99Tcm-MIBI parathyroid scintigraphy. ( 3 ) Surgical procedures: local or general anesthesia for PTX. Supplement with calcium and calcitriol implemented low serum calcium after PTX. (4) Follow-up: symptoms, including bone pain, muscle weakness, skin itching, and insomnia, were significantly improved after surgery. Transient hoarseness occurred in 2 cases. The iPTHs of all patients were decreased significantly after surgery. The median of iPTH was 55.5 ( 10-967) ng/L at 1 month post PTX, and was significantly less than prior to PTX (P＜0. 001). Eight patients were cure , 1 marked effectiveness ,and 1 ineffectiveness. Two patients were persistent SHPT, and 1 died of heart failure in the 4th year after PTX. The development of bone deformities was stopped and malnutrition was improved in long-time follow up. The level of iPTH 135(28-390)ng/L(P＜0. 001 ) , serum calcium, phosphorus, and ALP showed normal in the third year. The SHPT recurrence was appeared in the 2nd and 3rd year in 2 out of 8 patients, respectively. Conclusions Total PTX can effectively treat SS by SHPT. It can improve prognosis for patients, such as bone pain disappearing, bone deformities stopping and malnutrition improving, etc. The level of iPTH may rise again in some patients in the future. Therefore, more attentions should be paid to monitoring.

According to the physiological characteristics of lumbar vertebrae in Chinese, we designed and made a lumbar vertebral fusion cage of titanium and then engaged in its biomechanical test. T12-S1 of lumbar vertebrae from 18 fresh dead bodies were used. We measured the stress relaxation and the creep effects of the normal group (T12-S1 of intact lumbar vertebrae), the control group 1(simulating operation of excising intervertebral disc and planting bone on the back route) and the control group 2(simulating operation of excising intervertebral disc and inserting fusion cage). The data and stress, strain-time curves under the condition of constant stress and strain were obtained. Regression analysis yielded the reduced stress relaxation and creep functions. Finally, we analyzed and discussed the effects of the operation of excising intervertebral disc and planting bone on the back route and the operation of excising intervertebral disc and inserting fusion cage on the stability of spine.
Elasticity ; Humans ; Intervertebral Disc ; physiology ; surgery ; Lumbar Vertebrae ; physiology ; Spinal Fusion ; Spine ; Titanium

ObjectiveTo establish a novel assay for HIV-1 p24 ultrasensitive detection based on Gold Nanoparticle Probe (GNP) and PCR.MethodsSandwich ELISA method was established by a pair of anti-p24 monoclonal antibodies (mAbs),1G12 and 1D4,and was used to detect recombinant HIV-1 p24 antigen.The bio-barcode DNA was 47 bp,selected from genome of Arabidopsis,and formed double-stranded DNA by hybridization with the capture DNA (complementary with bio-barcode DNA) modified with sulfhydryl.Then double-stranded DNA were conjugated on the surface of 1D4-modified gold nanoparticles by sulfhydryl,and the Gold Nanoparticle Probe was produced.1G12 was precoated in the micropaltes,and in the presence of target recombinant HIV-1 p24 protein,a sandwich immuno-complex would form by adding GNP.Then the bio-barcode DNA in the immuno-complex were released by heating as detection signal,and consequently characterized by the polymerase chain reaction (PCR) with synthesized special primers and analyzed by 4％ agar gel electrophoresis,so HIV-1 p24 antigen could be evaluated.The sensitivity comparison between the new assay and ELISA can be done.ResultsSandwich ELISA was used to quantify HIV-1 p24 antigen by monoclonal antibodies 1G12 and 1D4,and the limit of detection (LOD) was 1000 pg/ml.The new GNP assay was established by the same pair of antibodies,combined with PCR and agar gel electrophoresis,and was used to indirectly detect HIV-1 p24 antigen.The band intensity of PCR products paralleled with the quantity of HIV-1 p24 antigen,and the limit of detection (LOD) could reach down to 1 pg/ml.ConclusionThe new assay based on GNP and PCR was efficient in the detection of HIV-1 p24,which is at least 3 orders of magnitude more sensitive than traditional ELISA.

Objective:To study the expression of methyltransferase-like protein 14 (METTL14) in epithelial ovarian cancer and its clinical significance, and to explore the effect of METTL14 expression on the proliferation, invasion and migration of ovarian cancer cells.Methods:Immunohistochemistry (IHC) was used to detect METTL14 expression in tumor tissue samples, and analyze the relationships among METTL14 expression, clinicopathological factors, and prognosis in ovarian cancer. Lentiviral vectors and small interfering RNA (siRNA) were used to up-regulate and down-regulate the METTL14 expression in ovarian cancer cell lines A2780 and SKOV3, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to detect the N6-methyladenosine (m6A) content in ovarian cancer cells. Cell counting kit-8 (CCK-8), wound healing assay, and transwell assay were used to examine the function of METTL14 expression in the cells.Results:(1) The IHC score of METTL14 protein was 6.2±3.7 in 20 samples of ovarian cancer tissues and 3.3±2.5 in 15 samples of normal ovarian tissues, and the difference was statistically significant ( t=-2.64, P=0.012). Among the patients who suffered from ovarian cancer, there were 69 cases with high expression of METTL14 protein (IHC score≥6), accounting for 57.0% (69/121), and the cases with low expression of METTL14 protein (IHC score<6) accounting for 43.0% (52/121). Compared with the patients with low expression of METTL14, the patients with high expression of METTL14 had later stages, higher rates of lymph node metastasis, abdominal metastasis, and more ascite amount. The differences were statistically significant (all P<0.05). The overall survival rate was significantly lower in patients with high METTL14 expression than the low expression ( P=0.009). (2) LC-MS/MS data showed that the relative expression of m6A in A2780 and SKOV3 cells in the lentivirus (LV)-METTL14 group were 0.213±0.024 and 0.181±0.018, which were significantly higher than those in the LV-normal control (NC) group (0.109±0.022 and 0.128±0.020; all P<0.05). While the relative expression of m6A in A2780 and SKOV3 cells in the si-METTL14 group were 0.063±0.012 and 0.069±0.015, which were significantly lower than the expression in si-NC group of 0.108±0.014 and 0.121±0.014 (all P<0.05). CCK-8 assay showed that the absorbance values were significantly lower in the si-METTL14 group compared with the si-NC group at 36, 48, 60 hours (all P<0.05); while were significantly increased in the LV-METTL14 group compared with the LV-NC group at 48, 60 hours (all P<0.01). Scratch wound assays showed that the migration rate of the si-METTL14 group was lower than those of the si-NC group, while the LV-METTL14 group were higher than the LV-NC group by 24 hours, the differences were statistically significant (all P<0.01). Cell migration and invasion were detected by transwell migration and invasion assays. After cultivated for 24 hours, the invasion cell number and the migration cell number in the si-METTL14 group were less than those in the si-NC group. While the invasion cell number and the migration cell number in the LV-METTL14 group were more than those in the LV-NC group, respectively. The differences were statistically significant (all P<0.01). Conclusion:Patients with high METTL14 expression have a worse prognosis in ovarian cancer, which may increase the m6A modification of ovarian cancer cells and promote cells proliferation, invasion and migration.

Neuropathic pain has been hypothesized to be the result of aberrant expression and function of sodium channels at the site of injury. To investigate the effects of NaV1.8 antisense oligonucleotide on the expression of sodium channel mRNA in dorsal root ganglion (DRG) neurons in chronic neuropathic pain. 24 Sprague-Dawley rats weighing 200-260 g were anesthetized with the in of sciatic nerve trunk by 4-0 chromic gut. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. A PE-10 catheter was implanted in subarachnoid space at lumbar region. On the 7th postoperative day the animals were randomly divided into 4 groups. The drugs were injected intrathecally twice a day for 5 consecutive days in group 2-4. The animals were decapitated 14 days after the surgery. The L4-L6 DRG of the operated side was removed and crushed, and total RNA was extracted with Trizol reagent. The contralateral side was used as control. The change of NaV1.8 sodium channel transcripts was determined by RT-PCR. Pain threshold was significantly lowered after CCI as compared with that in control group and was elevated 3 days after antisense oligonucleotide injection. Sensory neuron specific TTX-R sodium channel NaV1.8 transcript was down-regulated after antisense oligonucleotide injection at the dosage of 45 μg as compared with that in CCI group (P＜0.01), and it was even greater at the dosage of 90 μg. The intrathecally injected NaV1.8 antisense oligonucleotide can reduce the mechanical allodynia and thermal hyperalgesia partially by downregulating the SNS transcript expression.