OBJECTIVETo compare the changes of nucleus pulposus after in vitro culture of rabbit whole intervertebral disc and spinal motion segment.

METHODSTwenty-one New Zealand white rabbits which were randomly divided into organ group with 8 rabbits and segment group with 13 rabbits. Fifty intervertebral discs and 50 spinal motion segments were harvested respectively under aseptic conditions from two groups. These specimens were maintained in organ culture with hyperosmotic media (410 mOsm/kg), then 10 discs of the two groups were observed respectively by HE staining, immunohistochemistry of collagen type III, proteoglycan content and cells viability of nucleus pulposus before culture and at 3, 7, 14, 21 days after culture.

RESULTSHE staining showed the intervertebral disc tissue structure remained intact after culture of 21 days organ group and 14 days segment group,but there was severely degenerated of 21 days segment group. The intensity value of type II collagen immunohistochemical staining in the nucleus pulposus were not changed significantly between 21 days organ group and 14 days segment group (P > 0.05), but the staining of segment group at 21 days became shallower, there was significant difference compared with before each time points and organ group at 21 days (P < 0.05). PAS/AB staining of proteoglycan of nucleus pulposus showed that there were not decrease of tinting strength of two groups within 7 days, but the strength weakened slightly of two groups at 14 days, and the tinting strength became weaker at 21 days segment group, the change is more obvious than the organ group. The intensity value of fluorescence staining of nucleus pulposus cells was not changed significantly within 7 days of two groups (P > 0.05), the intensity value decreased slightly at 21 days organ group and 14 days segment group, but there were no significant difference compared with before time points (P > 0.05) however at 21 days segment group the intensity decreased as cells viability of nucleus pulposus decreased,and there was a significant difference compared with before each time points and organ group at 21 days (P < 0.05).

CONCLUSIONIt is not obviously degenerated of the discs of organ group cultured within 21 days and segment group cultured within 14 days, but there was significant degeneration of the intervertebral disc of segment group after cultured 21 days, so the rabbit spinal motion segment can be used on research about the biomechanics of intervertebral disc as a vitro experimental model within 14 days.

Animals ; Collagen Type II ; analysis ; Female ; Immunohistochemistry ; Intervertebral Disc ; chemistry ; pathology ; Male ; Organ Culture Techniques ; Rabbits

·AIM: To observe the effect of two kinds of bandage contact lenses on epithelial erosions and corneal thickness after vitreoretinal surgery for proliferative diabetic retinopathy ( PDR) .· METHODS: In this prospective, nonrandomized, comparative clinical study, 69 eyes of 69 patients with PDR were divided into two groups. They underwent vitreoretinal surgery. Group A, 36 eyes of 36 cases, the bandage contact lens with diameter of 13. 8mm were covered on corneal surface during surgery under noncontact wide-angle viewing systems. Group B, 33 eyes of 33 cases, the bandage contact lens with diameter of 14. 0mm were covered on corneal surface during the same surgery. Visual acuity, intraocular pressure, slit-lamp examination, corneal fluorescein sodium staining, count of corneal endothelium cells, measure of corneal thickness before and after operation were assessed.·RESULTS: Pre-operation, corneal fluorescein sodium staining positive rate was 42% in Group A and 42% in Group B (x2=0. 004, P=0. 949). At 1d after surgery, the positive rate of was 47% in Group A and 45% in Group B (x2=0. 022, P=0. 883). At 2d after surgery, the positive rate of was 44% in Group A and 45% in Group B ( x2 =0. 007, P=0. 933). At 3d after surgery, the positive rate of was 44% in Group A and 42% in Group B (x2=0. 029, P=0. 886). At 7d after surgery, the positive rate of was 42%in Group A and 39% in Group B (x2=0. 037, P=0. 848). Count of corneal endothelium cells showed no significant difference between Group A and Group B(P>0. 05). Count of corneal endothelium cells of Group A before surgery and at 7d after surgery were 2779. 25 ± 329. 55 /mm2 , 2777. 14±331. 17 /mm2, without significant difference (t=0. 551, P=0. 585);those of Group B were 2678. 61±335. 64/mm2 , 2672. 45 ± 336. 25 /mm2 , without significant difference(t = 1. 774, P = 0. 086). Measure of corneal thickness was 519. 25±23. 42μm before surgery and 542. 03± 25.94μm after surgery in Group A (t=-6.854, P<0.001). Measure of corneal thickness was 525. 64 ± 20. 97μm before surgery and 551. 33±27. 87μm after surgery in Group B (t=-7. 204, P<0. 001).·CONCLUSION:Two kinds of bandage contact lenses are used in vitreoretinal surgery in diabetic patients. The corneal epithelial integrity shows no difference before and after surgery. Both the bandage contact lens could protect the corneal epithelium and maintain good corneal transparency during vitreoretinal surgery.

Objective To investigate the role of overexpression of Smad7,the inhibitory factor of TGF-?/Smads signaling,in epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells.Methods Peritoneal fibrosis rat model was built by daily intraperitoneal injection with 4.25% Dineal (100 ml/kg) and lipopolysaccharide(LPS) (0.6 mg/kg) at day 8,10,12,22,24,26. Smad7 or control empty vectors was transferred at day 0,14 and was induced by doxycline in the daily drinking water (200 mg/L).Rats were sacrificed on day 28 and the expression of TGF-beta/ Smads,?-SMA and E-cadherin was examined.Results Compared with normal rats,empty vector rats showed higher expression of phosphorylated Smad2/3.?-SMA expression was elevated but E-cadherin was reduced.Under electron microscope,the mesothelial cells removed to submesothelial zone and showed large bundles of actin microfilaments and dense bodies within the cytoplasm. Basement membrane was broken.After induction of Smad7 in peritoneal fibrosis rats,the morphology of mesothelial ceils normalized partly,phosphorylated Smad2/3 was reduced.Moreover,expression of E-cadherin was increased,expression of?-SMA was dramatically reduced.Conclusion Inhibition of TGF-?/Smad signaling by Smad7 overexpression may inhibit the epithelial-mesenchymal transition of mesothelial cell,which may provide a new therapeutic method for peritoneal fibrosis by overexpression of Smad7.

As one of the main water-soluble composites of Radix Salviae, salvianolic acid B is a phenolic acid ingredient of the Chinese drug, which is rich content in the herb and has strong pharmaceutical activity. It is used to treat cardiocerebral vascular diseases, antagonize hepatic/renal fibrosis, prevent cancer, and promote stem cell proliferation and differentiation. In the researches of its acting mechanisms, rather deepened studies have been carried out for its application on cardiocerebral vascular diseases, but that for others are rather fewer.
Benzofurans ; pharmacology ; therapeutic use ; Biomedical Research ; trends ; Disease ; Humans ; Medicine, Chinese Traditional ; trends ; Stem Cells ; drug effects ; Ventricular Remodeling ; drug effects

Endoplasmic reticulum (ER) stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. The purpose of the present study was to investigate the effects of caveolin-1 (Cav-1) on ER stress-induced apoptosis in cultured macrophages and the underlying mechanisms. RAW264.7 cells were incubated with thapsigargin (TG) to establish ER stress model. And Cav-1 expression was detected by Western blot. After being pretreated with filipin(III), a caveolae inhibitor, RAW264.7 cells were assayed with flow cytometry and confocal laser scanning microscopy to detect cell apoptosis. Moreover, p38 mitogen-activated protein kinase (MAPK) phosphorylation and C/EBP homologous protein (CHOP) expression were detected with Western blot. The results showed that Cav-1 expression was markedly increased at early stage of TG treatment (P < 0.05) and then decreased with prolonged or high dose TG treatments. The increasing of Cav-1 expression induced by TG in RAW264.7 cells was abolished under inhibition of caveolae by filipin(III) (P < 0.05). The effect of TG on apoptosis of RAW264.7 cells was further augmented after pretreatment with filipin(III) (P < 0.05). Western blotting showed that MAPK phosphorylation induced by TG was inhibited by filipin(III) in RAW264.7 cells (P < 0.05), whereas CHOP remained unchanged (P > 0.05). These results suggest that Cav-1 may play a critical role in suppressing ER stress-induced macrophages apoptosis in vitro, and one of the mechanisms may be correlated with the activation of p38 MAPK prosurvival pathway.
Animals ; Apoptosis ; drug effects ; Caveolin 1 ; genetics ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; physiology ; Filipin ; pharmacology ; MAP Kinase Signaling System ; Macrophages ; cytology ; drug effects ; Mice ; Thapsigargin ; pharmacology ; Transcription Factor CHOP ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVEThis study was designed to detect the expression of bcl-2 and p53 proteins in colorectal carcinomas and to determine their association with the patient survival and stage of the diseases.

METHODSImmunohistochemistry method was used to detect the expression of bcl-2 and p53 proteins in 93 cases of colorectal carcinoma. The stain results were obtained by analyzing the clinic-pathological characteristics of patients.

RESULTSFifty-seven percent (53/93) of the colorectal carcinomas were bcl-2 protein positive. The positive rate of bcl-2 protein in lymph node involvement cases was lower (15/37) than the cases without node involvement (38/58, P<0.01). The positive rate of p53 protein was 43% (40/93) in colon-rectum carcinomas. No significant correlation was observed between p53 protein expression and clinic-pathological manifestations (P>0.05) but the survival was significantly worse (P=0.0001) in the p53 protein positive cases. Neither bcl-2 nor p53 alone was correlated with stage of the disease. When combined bcl-2/p53 status was analyzed, a group with bcl-2(+) and p53(-) had the best prognosis. This group was significantly associated with earlier Dukes' stages (P=0.1763). In multivariate Cox regression analysis, lymph node involvement and p53 protein expression were two independent factors correlated with survival time.

CONCLUSIONThe expression of bcl-2 and p53 represent biological characteristics of colorectal carcinomas. Assessment of both bcl-2 and p53 status may be valuable in predicting the prognosis of patients.

Biomarkers, Tumor ; metabolism ; China ; epidemiology ; Colorectal Neoplasms ; diagnosis ; metabolism ; mortality ; Female ; Humans ; Male ; Middle Aged ; Prevalence ; Prognosis ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Risk Assessment ; methods ; Risk Factors ; Survival Analysis ; Survival Rate ; Tumor Suppressor Protein p53 ; metabolism

AIMTo study the distribution and efficiency of pEGFP-N1 plasmid after its transfection in cardiomyocytes.

METHODSThe neonatal rat cardiomyocytes were cultured. According to the different grow period of neonatal rat cardiomyocytes, the distribution and efficiency of pEGFP-N1 plasmid after its transfection in cardiomyocytes were studied.

RESULTSThe efficiency was significantly increased after pEGFP-N1 plasmid transfection in one-day cardiomyocytes. The distribution of EGFP was in the cytoplasm and nucleus.

CONCLUSIONThe efficiency of pEGFP-N1 plasmid after its transfection in cardiomyocytes was related to the grow period of neonatal rat cardiomyocytes. The distribution of EGFP was in the cytoplasm and nucleus.

Animals ; Animals, Newborn ; Cells, Cultured ; Green Fluorescent Proteins ; genetics ; metabolism ; Myocytes, Cardiac ; metabolism ; Plasmids ; Rats ; Rats, Wistar ; Transfection

A high performance liquid chromatography (HPLC) method was developed for fingerprint of Dipsacus asper. Analysis were carried out on a Zorbax C-18 column by gradient elution using 0.1% phosphoric acid and acetonitrile as the mobile phases. The column was maintained at 25 degrees C, the flow rate was 1 mL x min(-1), and the detection wavelength was set at 205 nm. Asperosaponin VI was selected as reference compound, Seventeen common peaks were selected, and the fingerprint with good precision, stability and repeatability was successfully used to evaluate quality of 24 batches of crude extracts of D. asper. Chemical characteristics of D. asper was analyzed by DAD detection and HPLC-MS techniques with an ESI source. The quasi-molecular ions of compounds in both negative and positive modes were observed for molecule mass information of 33 compounds, and the potential structures of 10 characteristic components were identified by study on the mass spectra of compounds and comparing with reference data and some of standards. The results indicate the HPLC fingerprint of D. asper will show more characters through identification of component structures using an HPLC-ESI-MS method, and will control the quality of D. asper more effectively and reasonable.
Chromatography, High Pressure Liquid ; methods ; Dipsacaceae ; chemistry ; Drugs, Chinese Herbal ; analysis ; isolation & purification ; Spectrometry, Mass, Electrospray Ionization ; methods

BACKGROUNDThe most significant biological change in intervertebral disc degeneration is the decrease of chondrocyte specific gene and protein expression of Sox9 and collagen type II. Interleukin-1 (IL-1) is not expressed in the normal intervertebral disc tissue but increases in the degenerated intervertebral disc tissue. This suggests that IL-1 may play a role in regulation of the expression of Sox9 and collagen type II.

METHODSHuman intervertebral disc cells were isolated and cultured. Sox9 and collagen type II expression during treatment with IL-1, with or without the nuclear factor-kappaB (NF-kappaB) activity inhibitor curcumin, were detected by using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the activity of the NF-kappaB signaling pathway was detected by the electrophoretic mobility shift assay (EMSA).

RESULTSIL-1 lowered the mRNA level and protein expression of Sox9 and collagen type II in the cultured intervertebral disc cells in a dose dependent manner (P < 0.05), and this effect was attenuated by curcumin. Curcumin alone had no effect on Sox9 and collagen type II expression (P > 0.05). IL-1 at concentrations of 0.1 ng/ml, 1 ng/ml and 10 ng/ml could stimulate the activity of NF-kappaB in the intervertebral disc cells in a dose dependent manner (P < 0.05) that was inhibited by curcumin.

CONCLUSIONSWe demonstrated the previously unknown function of IL-1 in inhibiting Sox9 and collagen type II via NF-kappaB in the intervertebral disc cells. This inhibition can be attenuated by curcumin, which is an effective NF-kappaB activity inhibitor.

Adult ; Cells, Cultured ; Collagen Type II ; genetics ; metabolism ; Curcumin ; pharmacology ; Electrophoretic Mobility Shift Assay ; Gene Expression ; drug effects ; Humans ; Immunoblotting ; Interleukin-1 ; pharmacology ; Intervertebral Disc ; cytology ; Male ; NF-kappa B ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; SOX9 Transcription Factor ; genetics ; metabolism

OBJECTIVETo study the therapeutic efficacy of siRNA fragments silencing p75 neurotrophin receptor (p75(NTR)), which may be a key regulator of glioma cell apoptosis and invasion.

METHODSThe siRNA sequence fragments targeting p75(NTR) were designed and transferred into human glioma cell line U251. RT-PCR and immunocytochemistry method were used to explore the expression of p75(NTR) mRNA and protein. Cell adhesion assay was employed to detect cellular adhesion ability, and soft agar clone formation assay was adopted to identify oncogenicity, and a U251 glioma model was established in nude mice. The intracranial tumor volume was detected by MRI. The expression of p75(NTR), NGF and cyclin D2 were identified using immunohistochemistry. Cell apoptosis was detected by apoptosis kit in situ.

RESULTSThe siRNA fragments targeting p75(NTR) were capable of decreasing mRNA and protein expression of p75(NTR) in U251 glioma cell line. Both the cellular adhesion ability and oncogenicity were weakly relevant. The p75(NTR) expression level was negatively correlated with cyclin D2 and apoptosis, and positively correlated with NGF expression. The siRNA sequence fragments targeting p75(NTR) were effective in decreasing the gross volume of tumor; prolonged the survival time of mice, and the edge of tumor was much sharper than that of the control group.

CONCLUSIONSThe gene silencing technique by siRNA targeting p75(NTR) is capable of decreasing tumor invasion and cell proliferation as well as inducing cell apoptosis. It is expected to be a new choice for glioma gene therapy.

Animals ; Apoptosis ; Brain Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin D2 ; metabolism ; Gene Silencing ; Glioma ; genetics ; metabolism ; pathology ; Humans ; Male ; Mice ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Nerve Growth Factor ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Random Allocation ; Receptor, Nerve Growth Factor ; genetics ; metabolism