[Clinical study of application minimally invasive expandable channel in lumbar discectomy and interbody fusion and internal fixation].

OBJECTIVE: To explore the advantages of minimally invasive expandable in surgery of lumbar discectomy and interbody fusion and internal fixation. METHODS: The clinical data of 48 patients who underwent lumbar discectomy and interbody fusion and internal fixation from January 2010 to March 2016 was retrospectively analyzed. According to the admission queue, the patients were randomly assigned into channel group (26 cases) or traditional group (22 cases). In channel group, surgical approach of minimally invasive expandable channel was applied, and in traditional group, open posterior operation approach (including posterior lumbar interbody fusion and transforaminal lumbar interbody fusion, etc.) was applied. In channel group, there were 20 males and 6 females, aged from 43 to 74 years with an average of(56.6±5.1) years; course of disease was ranged from 4 to 22 months with an average of (6.7±1.8) months; 1 case was complicated with diabetes, 6 cases were complicated with hypertensive disease, and 2 cases were complicated with arrhythmia. In traditional group, there were 15 males and 7 females, aged from 43 to 73 years with an average of(55.9±4.6) years; course of disease was ranged from 4 to 26 months with an average of (6.2±2.1) months; 2 cases were complicated with diabetes, 5 cases were complicated with hypertensive disease, and 1 case was complicated with arrhythmia. Operation time, bleeding volume, and hospitalization time were compared between two groups and visual analogue scale(VAS), Oswestry Disability Index(ODI), bone fusion information, and complications correlated with incision were observed in two groups. RESULTS: All 48 patients were followed up for more than 6 months. Postoperative VAS and ODI were significantly improved (P<0.01), but 3 and 6 months after operation, there was no significant difference in VAS between two groups, and ODI score of channel group was lower than that of traditional group(P<0.01). Operation time, bleeding volume, hospitalization time in channel group respectively were (167.3±30.2) min, (786.8±147.8) ml, (12.3±2.4) d, and in traditional group were (197.5±48.7) min, (786.8±147.8) ml, (16.5±3.8) d, there was significant differences between two groups. There was no significant difference in fusion rate and fusion time between two groups. There were 4 cases and 7 cases developed incision related complications in channel group and traditional group, respectively. The difference between two groups was significant(P<0.01). CONCLUSIONS: Compared with conventional surgery minimally invasive lumbar discectomy and interbody fusion and internal fixation has advantages of less trauma, shorter operative time and better functional recovery.

Potential Role of Hepatitis C Virus Alternate Reading Frame Protein in Negative Regulation of T-Bet Gene Expression.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and has led to cirrhosis or hepatocellular carcinoma in a majority of infected individuals. We have previously demonstrated that the HCV alternate reading frame protein (F protein) is related to Th1/Th2 bias in chronic hepatitis C (CHC) patients, and we aimed to explore the relative molecular mechanisms here. A total of 104 cases including CHC patients and healthy donors were enrolled. T-bet and GATA-3 expression levels were analyzed in peripheral blood mononuclear cells (PBMCs). The levels of signal transducer and activator of transcription-1/-6(STAT1/6) and phosphorylated STAT1/6(pSTAT1/6) in PBMCs were measured by Western blotting. Our results showed that the levels of T-bet in PBMCs, as well as the levels of gamma interferon (IFN-γ) in sera, were decreased in anti-F protein antibody seropositive patients compared with anti-F protein antibody seronegative patients, whereas the levels of GATA-3 did not show difference between the two groups. Moreover, the decreased pSTAT1 and increased pSTAT6 were observed in PBMCs by HCV core/F protein stimulation with constant STAT1/6 expression. Taken together, it suggested that T-bet may be involved in Th1/Th2 bias induced by HCV F protein, and the disruption of STAT phosphorylation may participate in this mediation.

Dendritic development of hippocampal CA1 pyramidal cells in a neonatal hypoxia-ischemia injury model.

It is believed that neonatal hypoxia-ischemia (HI) brain injury causes neuron loss and brain functional defects. However, the effect of HI brain injury on dendritic development of the remaining pyramidal cells of the hippocampus and the reaction of contralateral hippocampal neurons require further studies. The Morris water maze and Golgi-Cox staining were used to evaluate the learning and memory and dendritic morphology of pyramidal cells. The results of Golgi-Cox staining showed CA1 pyramidal neurons of HI injury models with fewer bifurcations and shorter dendrite length than the naive control group. The density of dendritic spines of hippocampal CA1 pyramidal neurons was significantly lower in the HI brain injury group than in controls. With respect to hippocampal function, the HI brain injury group presented cognitive deficits in the reference memory task and probe trail. In the HI group, the pyramidal cells of left hippocampus that did not experienced ischemia but did experience hypoxia had more complex dendrites and higher density of spine than the HI injury side and control. The functional implementation of injured hippocampus might depend mainly on the hypertrophy of contralateral hippocampus after HI brain injury. Corticosterone can partially prevent the hippocampal pyramidal cells from HI injury and reduce the difference of the bilateral hippocampus pyramidal cells, but there was no improvement in learning and memory.

[Effects of biochars produced from different sources on arsenic adsorption and desorption in soil].

By using OECD Guideline 106 batch equilibrium method, this paper studied the characteristics of As (V) adsorption and desorption in brown soil as affected by the biochars produced from dairy manure, pine needle, and corn straw. When the addition amount of the biochars was 0.5%, the maximum adsorption amount of As (V) was decreased in the order of dairy manure biochar > pine needle biochar > corn straw biochar, which was related to the basic characteristics of the biochars. The adsorption isotherm of As (V) could be well fitted by Langmuir model (R2 = 0.997). In comparing with CK, both the adsorption capacity (lgKf = 1.99-2.10) and the adsorption intensity (1/N = 0.413-0.449) of As (V) were low, and the main adsorption mechanism was physical adsorption. The desorption rate of As (V) (14.5%-18.7%) was decreased in the order of dairy manure biochar > pine needle biochar > corn straw biochar. The addition of the biochars decreased the adsorption of As (V) by brown soil, which could induce the increase of the bioavailability of As, and strengthen the toxicity of As in soil.

Expression of a dominant-negative Rho-kinase promotes neurite outgrowth in a microenvironment mimicking injured central nervous system.

AIM: To investigate whether lentiviral vector (LV)-mediated expression of a dominant negative mutant Rho-kinase (DNROCK) could inhibit activation of the Rho/ROCK signaling pathway and promote neurite outgrowth in a hostile microenvironment mimicking the injured central nervous system (CNS) in vitro. METHODS: Lentiviral stock was produced using the three-plasmid system by transfecting HEK293 cells. Myelin prepared from rat brain was purified by two rounds of discontinuous density gradient centrifugation and osmotic disintegration. Differentiated PC12 cells and dissociated adult rat dorsal root ganglion (DRG) neurons were transduced with either LV/DNROCK or LV/green fluorescent protein (GFP) and seeded on solubilized myelin proteins. The effect of DNROCK on growth cone morphology was tested by rhodamine-conjugated phalloidin staining. Expression of DNROCK was determined by immunoblotting. The length of the longest neurite, the percentage of neurite-bearing neurons, or the total process outgrowth for all transduced neurons were measured by using the Scion image analysis program. RESULTS: Transduction of DNROCK inhibited serum-induced stress fiber formation in NIH 3T3 cells and induced enlargement of cell bodies and decreased the phosphorylation levels of MYPT1 in HeLa cells. LV/DNROCK blocked myelin-induced increase in ROCK translocation from cytosol to membrane in LV/GFP-treated PC12 cells. DNROCK promotes neurite outgrowth of differentiated PC12 cells and DRG neurons on myelin protein. LV/DNROCK-transduced PC12 cells had longer neurites than LV/GFP-transduced cells (39.18+/-2.19 microm vs 29.32+/-1.7 microm, P<0.01) on myelin-coated coverslips. Furthermore, a significantly higher percentage of LV/DNROCK-transduced cells had extended neurites than LV/GFP-transduced cells (63.75%+/-8.03% vs 16.3%+/-3.70%, P<0.01). LV/DNROCK-transduced DRG neurons had longer neurite length (325.22+/-10.8 microm vs 202.47+/-9.3 microm, P<0.01) and more primary neurites per cell than those in LV/GFP-transduced cells plated on myelin and laminin (7.8+/-1.25 vs 4.84+/-1.45, P<0.01) or on laminin alone (5.2+/-1.88). LV/DNROCK-transduced cells had significantly larger growth cones (33.12+/-1.06 microm(2)) than LV/GFP-pretreated cells (23.72+/-1.22 microm(2)). CONCLUSION: These results indicate that blocking the RhoA/ROCK signaling pathway by expression of DNROCK is effective in facilitating neurite outgrowth in a microenvironment mimicking injury of central nervous system.

Inhibition of ATP-induced glutamate release by MRS2179 in cultured dorsal spinal cord astrocytes.

It was reported that ATP, an excitatory chemical mediator, exerts its effects by activation of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) purinoceptors in the nervous system. In the present work, we used confocal laser scanning microscopy and high-performance liquid chromatography to assess the role of the P2Y1 receptor in ATP-evoked Ca2+ mobilization and glutamate release from cultured dorsal spinal cord astrocytes. ATP (0.01-100 micromol/l) produces a dose-dependent rise in the Ca2+ relative fluorescence intensity in cultured astrocytes. N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179, 0.01-100 micromol/l), a P2Y1-specific antagonist, could dose-dependently inhibit ATP-evoked Ca2+ mobilization. In addition, 100 micromol/l ATP caused glutamate efflux from cultured dorsal spinal cord astrocytes in a time-dependent manner. 100 micromol/l MRS2179 significantly inhibited the glutamate efflux induced by ATP, which suggests that P2Y1 receptor activation is responsible for the ATP-induced glutamate efflux from astrocytes. Taken together, our results demonstrate that P2Y1 receptor plays an important role in modulating the function of astrocytes, which raises the possibility that MRS2179, a potent P2Y1-specific antagonist, may become a potential drug in treating many chronic neurological diseases characterized by astrocytic activation in the nervous system.

P2Y1 receptor-mediated glutamate release from cultured dorsal spinal cord astrocytes.

P2 receptors have been implicated in the release of neurotransmitter and proinflammatory cytokines by the response to neuroexcitatory substances in astrocytes. In the present study, we examined the mechanisms of ADP and adenosine 5'-O-2-thiodiphosphate (ADPbetaS, ADP analogue) on glutamate release from cultured dorsal spinal cord astrocytes by using confocal laser scanning microscopy and HPLC. Immunofluorescence activity showed that P2Y(1) receptor protein is expressed in cultured astrocytes. ADP and ADPbetaS-induced [Ca(2+)](i) increase and glutamate release are mediated by P2Y(1) receptor. Ca(2+) release from IP(3)-sensitive calcium stores and protein kinase C (PKC) activation is important for glutamate release from astrocytes. Furthermore, P2Y(1) receptor-evoked glutamate release is regulated by volume-sensitive Cl(-) channels and anion co-transporter, which open up the possibility that P2Y(1) receptor activation causes the increase of cell volume. Release of glutamate by ADPbetaS was abolished by 5-nitro-2 (3-phenyl propy lamino)-benzoate plus furosemide but was unaffected by botulinum toxin A. These observations indicate that P2Y(1) receptor-evoked glutamate may be mediated via volume-sensitive Cl(-) channel but not via exocytosis of glutamate containing vesicles. We speculate that P2Y(1) receptors-evoked glutamate efflux, occurring under pathological condition, may modulate the activity of synapses in spinal cord.

[Progress of studies on multidrug resistance in hematological malignancies--review].

Chemotherapy remains a major route of intervention in hematological malignancies. However, a key issue in the treatment of hematological malignancies is the development of multidrug resistance (MDR) to chemotherapeutic drugs. Several mechanisms may account for this phenomenon, including biochemical mechanisms, such as the overexpression of resistance-conferring proteins and physiological mechanisms involving the hematopoietic microenvironment. In this article the pathomechanism, diagnostic approach, interpretation of results from clinical samples and correlations with hemopoietic microenvironment were briefly reviewed. The aspects of development and problems in MDR study as well as detection methods for MDR were also discussed.

[Construction and expression of adeno-associated virus vectors of Smad 6 and Smad 7 genes in human renal tubule epithelial cells].

AIM: To construct the adeno-associated virus(AAV) vectors of Smad 6 and Smad 7 genes and observe their expressions in human renal tubule epithelial cells. METHODS: The plasmids pcDNA3-Smad 6/flag and pcDNA3-Smad 7/flag were digested with BamH I and Xho I. Then the Smad 6/flag and Smad 7/flag gene fragments were cloned into plasmid pAAV-MCS, respectively to construct the recombinant pAAV-Smad 6/flag and pAAV-Smad 7/flag plasmids. The recombinant expression plasmid or pAAV-LacZ plasmid were co-transfected into the HEK 293 cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect human renal tubule epithelial cells (HKCs), The expressions of Smad 6 and Smad 7 in HKCs were demonstrated by immunocytochemical staining. RESULTS: The recombinant AAV vectors of Smad 6 or Smad 7 genes were constructed and expressed in the HKCs successfully. CONCLUSION: These results indicate that AAV can deliver Smad 6 and Smad 7 genes to renal cells in-vitro, suggesting the recombinant AAV can be used for gene therapy of renal fibrosis.

[Detection of Schistosomia japonicum 5D gene by polymerase chain reaction and genechip technique].

OBJECTIVE: In order to develop the diagnostic genechip for specific detection of Schistosoma japonicum (Chinese mainland strain). METHODS: Probe and primers were designed based on the Schistosoma japonicum 5D gene encoding an immunogenic miracidial antigen. The probe for the conservative and specific gene sequence was spotted onto the specially treated glass slides by pin-based spotting robot Pixsys 5500 and was employed to make genechips. A polymerase chain reaction (PCR) protocol was designed to effectively amplify the 5D gene fragment containing the probe sequence from cercaria, egg, adult worm and infected Oncomelania DNA as well as other flukes DNA, respectively. After 35 cycles by PCR, the products were then labeled with fluorescent Cy3-labeled primer, using dissymmetrical PCR. The labeled PCR products of the target genes were hybridized to the diagnostic genechips for detection of Schistosoma japonicum and a fluorescent scanner (ScanArray 3000) was used to observe and record the hybridization signals. RESULTS: The result obtained from the study showed that a 262 bp DNA fragment was amplified from cercaria, egg and adult worm with the designed primers and enable the genechip be applied to detect a single cercaria, egg and adult worm. When the genechip was used to detect Clonorchis sinensis, Fasciolopsis busk, and Paragonimus westermani DNA, the results showed negative, indicating that the genechip had good specificity. CONCLUSION: The genchip technique for detection of Schistosoma japonicum was established successfully and having the characteristics of high sensitivity and specificity.

Effect of antisense oligonucleotide of noggin on spatial learning and memory of rats.

AIM: To investigate the effect of antisense oligonucleotide (ASODN) of noggin on rat spatial learning and memory. METHODS: Expression of noggin mRNA was measured by in situ hybridization method and the ability to spatial learning and memory was tested with Morris water maze. RESULTS: Compared with control rats, noggin mRNA positive neurons in dentate gyrus (DG) and CA3 region of hippocampus were markedly increased after the Morris water maze training (P<0.01). The increase of noggin mRNA positive neurons in hippocampus following maze training could be significantly blocked by icv injection of antisense noggin ODN, and the injection also impaired the learning and memory formation as compared to that in control rats. But the sense oligonucleotide (SODN) had no effect. CONCLUSION: Noggin, as an embryonic gene expressed in adult hippocampus, plays an important role in the process of learning and memory formation.

Expression of an antitumor-analgesic peptide from the venom of Chinese scorpion Buthus martensii karsch in Escherichia coli.

The gene encoding a putative mature antitumor-analgesic peptide (AGAP) from the venom of the Chinese scorpion Buthus martensii Karsch was obtained by polymerase chain reaction (PCR) according to its cDNA sequence and expressed in Escherichia coli. While most of the recombinant AGAP was expressed in the form of insoluble inclusion body. The recombinant AGAP was purified to homogeneity by metal chelating affinity chromatography. Pharmaceutical tests showed that the recombinant AGAP has both analgesic and antitumor activities on mice.

[Expression, purification and serological reactivity of a chimeric antigen of GRA6 with P30 from Toxoplasma gondii].

Major surface protein (p30) and Dense Granule Antigen GRA6 of Toxoplasma gondii have good antigenicity, and could be used for detection of IgM against Toxoplasma gondii. GRA6 may complement P30 to reach more high sensitivity for detection of antibodies to Toxoplasma gondii, so, we try to express the chimeric protein of GRA6 and P30 by genetic engineering, identify its antignenicity and use for developing diagnosis reagent. Antigenic domains of p30 and GRA6 of Toxoplasma gondii were screened by analyzing their sequences using the software ANTHEWIN. Two DNA fragments encoding respectively antigenic domains of p30 and GRA6 were cloned, they were inserted into the same expression vector pET28a( + ) and expressed as a chimeric protein in Escherichia coli. BL21(DE3), the expressed chimeric protein of p30 with GRA6 in a form of inclusion body was about 25% of total proteins of E. coli. BL21(DE3). The inclusion body was washed once with 0.5% Triton X-100 and dissolved with 0.5% SKL, after renaturation by gradient dialysis, the recombinant protein was purified by DEAE-Sepharose FF cation column and then detected with 12% SDS-PAGE, it exists mainly in the eluted peak with 300 mmol/L NaCl and has high purity. By using enzyme-linked immunosorbent assay (ELISA), the recombinant protein was examined for reactivity with immunoglobulin M (IgM) antibodies in 6 sera from patients infected with Toxoplasma gondii ., it was reactive with all the 6 sera but not with sera from normal people, these results showed that the recombinant chimeric antigen has good antigenicity and specificity and could be used for detection of IgM against Toxoplasma gondii. The expressed chimeric protein could be used for epidemic investigation of Toxoplasma gondii, blood donor screening, especially for detection of pregnant women, and is of great significance in prevention of Toxoplasma gondii infection.