Evaluation of CD24 as a marker to rapidly define the mesenchymal stem cell phenotype and its differentiation in human nucleus pulposus

Background Recent studies have indicated that human nucleus pulposus contain mesenchymal stem cells （NP-MSCs）. However, the immunophenotypic variation of NP-MSCs in vitro was unclear. The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell. Methods Tissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures. Flow cytometric and laser scanning confocal microscope （LSCM） were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor [31 （TGF-131）. Results More than 90% of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs, such as CD44, CD105 and CD29, but the marker of mature NP cells characterized as CD24 was negative. In continuous cultures, the proportion of mesenchymal stem cells which were expressing CD44, CD105 and CD29 in nucleus pulposus gradually decreased. The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29, with slight positivity for CD44. The CD24 expression gradually increased in proliferation. Bi- parametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently, and only a small part of cells expressed both CD105 and CD24 simultaneously. TGF-{31 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24. Conclusions Non-degenerative and degenerative NP contains mesechymal stem cells. The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.     

Transplantation of gene-modified nucleus pulposus cells reverses rabbit intervertebral disc degeneration

Background  Intervertebral disc degeneration is the main cause of low back pain. The purpose of this study was to explore potential methods for reversing the degeneration of lumbar intervertebral discs by transplantation of gene-modified nucleus pulposus cells into rabbit degenerative lumbar intervertebral discs after transfecting rabbit nucleus pulposus cells with adeno-associated virus 2 (AAV2)-mediated connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinases 1 (TIMP1) genes in vitro.

Methods  Computer tomography (CT)-guided percutaneous annulus fibrosus injury was performed to build degenerative lumbar intervertebral disc models in 60 New Zealand white rabbits. rAAV2-CTGF-IRES-TIMP1-transfected rabbit nucleus pulposus cells were transplanted into degenerative lumbar intervertebral discs (transplantation group), phosphate-buffered saline (PBS) was injected into degenerative lumbar intervertebral discs (degeneration control group) and normal lumbar intervertebral discs served as a blank control group. After 6, 10 and 14 weeks, the disc height index (DHI) and signal intensity in intervertebral discs were observed by X-ray and magnetic resonance imaging (MRI) analysis. The expression of CTGF and TIMP1 in nucleus pulposus tissue was determined by Western blotting analysis, the synthesis efficiency of proteoglycan was determined by a ³⁵S-sulfate incorporation assay, and the mRNA expression of type II collagen and proteoglycan was detected by RT-PCR.

Results  MRI confirmed that degenerative intervertebral discs appeared two weeks after percutaneous puncture. Transgenic nucleus pulposus cell transplantation could retard the rapid deterioration of the DHI. MRI indicated that degenerative intervertebral discs were relieved in the transplantation group compared with the degeneration control group. The expression of collagen II mRNA and proteoglycan mRNA was significantly higher in the transplantation group and the blank control group compared with the degeneration control group (P <0.05).

Conclusions  CT-guided percutaneous puncture can successfully build rabbit degenerative intervertebral disc models. Both CTGF and TIMP1-transfected cell transplantation helps to maintain disc height, and promotes the biosynthesis of type II collagen and proteoglycan in intervertebral discs, reversing the degeneration of intervertebral discs.

     

Normal and degenerated rabbit nucleus pulposus cells in in vitro cultures: A biological comparison

This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type Ⅱ collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at G l phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G 1 phase and failed to enter S phase. The expression of type Ⅱ collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.     

The study of differences of antigenicity in rabbit intervertebral disc

<正>Objective To reveal and compare the immunogenicity of glycoprotein from nucleus pulposus and collagen from anulus fibrosus in rabbit intervertebral disc.Methods According to the similarity principle of genome biology and the amount of     

Effects of Chinese Herbal Medicine Serum on the Apoptosis of Sinoatrial Node Cells Induced by Simulated Ischemia-reperfusion

Objective:To study the effect of Chinese herbal medicine Kangxin Fumai Granule(康心复脉颗粒 Granule for heart diseases) serum on the primary cultured sinoatrial node(SAN) cell apoptosis induced by simulated ischemia-reperfusion(IR).Methods:The SAN cells removed from SAN tissue of neonatal Wistar rats were cultured and purified with differential attachment and 5’-bromodeoxyuridine(BrdU) treatment.Simulated IR model was adopted.The obtained cells were morphologically observed with inverted microscopy.By using the method of serum pharmacology,the cell apoptosis was measured with TUNEL staining qualitatively and with flow cytometry quantitatively.Results:Three kinds of cells were observed in the cultured SAN cells:spindle,triangle and irregular.The spindle cells comprised the greatest proportion.The SAN cells in the model group showed moderate positive brown staining in the nucleus,and the apoptosis rate increased significantly compared to that in the control group(P<0.01).While the SAN cells in the Kangxin Fumai Granule high-dose group did not demonstrated positive staining in the nucleus,and the apoptosis rate decreased significantly compared to that in the model group(P<0.05).Conclusion:Of the cells cultured from SAN,the spindle cells were pacemaker cells of SAN in rats.Blockade and/or inhibition of the SAN cell apoptosis might be one of the important mechanisms of Kangxin Fumai Granule in preventing and treating sinoatrial injury induced by simulated IR.     

Cerebellar Projections of the Pontine and Tegmento-reticular Ncleus in the Rabbit-An HRP Study

Total 19 adult healthy rabbits of both sexes weighing1.5～2.5kg are used in this study.A minute amount of 50% HRP wasinjected into the various lobes of the cerebellum.After the survival,perfusion and fixation,the brain stem together with the cerebellum wascut into transverse serial sections on a freezing microtome.The sectionswere incubated with DAB,BHDC or TMB.The labeled cells in thepontine nucleus and tegmento-reticular nucleus were investigated underbright-and darkfield microscope.The results are as follows:Following a single injection into the paramedian lobule,paraflocculusor the lateral part of the anterior lobe,the labeled neurons are found inthe bilateral dorsolateral,ventral,lateral and medial pontine subnuclei.After the injection into the posterior vermis,the labels are bilaterallypresented in the lateral and medial pontine subnuclei.The flocculusinjected with HRP shows labels in the dorsolateral and medial pontinesubnuclei and ansiform lobule receiving HRP shows the labeled cells inthe dorsolateral and ventral pontine subnuclei.The labeled cells arelocated in the rostral part of these subnuclei.In all the above mentioned cases,the distribution of the labeled cellsin the tegmento-reticular nucleus is organized without a distincttopography.These pontocerebellar projections are considered with regard totransmitting the visual and auditory and other information to thecerebellum.     

Relationship between the characteristics of immunopathological expression of hepatitis C virus antigen and hepatocytic injury.

Biopsied liver tissues from 352 cases were tested for hepatitis C virus (HCVAg) with improved PAP immunohistologic chemical method. Furthermore, corresponding seroantibody to hepatitis C virus was also tested. The total HCVAg positive rate was 9.1%. The HCVAg positive rate in chronic persistent hepatitis (CPH) was 5%. The HCVAg positive rate in chronic active hepatitis (CAH) was 11.2%. The HCVAg positive rate raised gradually along with the severity of hepatocytic injury. HCVAg may be seen in necrotic liver cells exfoliating into the liver sinus, indicating a close relationship between HCVAg and hepatocytic injury. Expression of HCVAg was mostly of the nucleus type in CPH cases and was mostly of the plasma type in CAH cases. The periphery of nucleus type-expressed positive cells generally had no marked inflammatory cell infiltration. The periphery of plasma type-expressed positive cells had a certain amount of inflammatory cell infiltration. Along with the severity of hepatocytic injury, HCVAg expressed itself in a positive correlation according to the nucleus and plasma types. The HCVAg positive cells were located mostly in the lobular peripheral band and rarely located in the venoperipheral band. It was possible that this had some relation with the lobular microcirculation of blood and blood supply. In this study, there was no obvious correlation between the HCVAg positive rate in hepatic tissues and the anti-HCV positive rate in sera. Neither the patients with HCVAg positive liver tissues nor the patients with seropositive anti-HCV had any history of blood transfusion and the use of blood products.(ABSTRACT TRUNCATED AT 250 WORDS)

     

The immunocytochemical localization of insulin-like growth factor Ⅱ in human cirrhosis

Sections of 30 cases of human cirrhosis were stained with rabbit anti-insulin-likegrowth factor Ⅱ(IGF Ⅱ)by double PAP method.By the serological examination 15 patientsshowed HBV infection and sections of 14 eases were HBsAg postively with a total rate of 67％(20 cases).The IGF Ⅱ was positive in the cytoplasm of all the liver and ductular cells.Binucle-ated,polypoid liver cells and the peripheral cells of the lobules or nodules were distinctly posi-tive,The liver cells which were strongly positive were a kind of thin polygonal cells with asmall oval or a round deeply stained nucleus in each.They might exist sporadically in the lob-ules or in the marginal portion of a nodule.These liver cells are quite different from the so-called oval cells which are derived from the proliferating ductules and are generally believed tobe responsible for the pathogensis of hepatoma.     

An animal model to create intervertebral disc degeneration characterized by radiography and molecular biology

Objectives:To develop a rabbit model of intervertebral disc degeneration that more exactly simulates the pathological changes of human intervertebral disc degeneration.Methods:Twelve New Zealand white rabbits were utilized to establish three different disc injury models according to the following protocol;group A:anulus punctures were done with a 18-gauge needle at L2-L3 and L5-L6; Group B:intradiscal injection of interleukin-1 IL-1βwith a 23-gauge needle at L3-L4;and Group C:intradiscal injection of phosphate buffer saline(PBS)with a 23-gauge needle at L4-L5.The L1-L2 level was used as a control.Rabbits were killed after 24 weeks.The intervertebral disc height was measured by lateral plain radiographs.After the radiographic measurements were obtained,the interver- tebral discs were removed and analyzed for DNA,sulfated glycosaminoglycan(s-GAG)and water contents of nucleus pulposus.Results: The intervertebral disc height,s-GAG,and water contents in anulus needle punctures were significantly decreased in Group A,but the DNA content in the nucleus pulposus was significantly increased when compared to the control.The significant decrease of disc height and water contents were demonstrated,only the s-GAG and DNA contents did not show a significant difference in Group B when compared to the control.The significant decrease of disc height,s-GAG,water,and DNA contents did not show in Group C when compared to the control.Conclusion:The 18-gauge puncture models produced the most consistent disc degeneration in the rabbit lumbar spine.     

An animal model to create intervertebral disc degeneration characterized by radiography and molecular biology

Objectives： To develop a rabbit model of intervertebral disc degeneration that more exactly simulates the pathological changes of human intervertebral disc degeneration. Methods： Twelve New Zealand white rabbits were utilized to establish three different disc injury models according to the following protocol; group A： anulus punctures were done with a 18-gauge needle at L2-L3 and L5-L6; Group B： intradiscal injection of interleukin-1 IL-1β with a 23-gauge needle at L3-L4; and Group C： intradiscal injection of phosphate buffer saline（PBS） with a 23-gauge needle at L4-LS. The L1-L2 level was used as a control. Rabbits were killed after 24 weeks. The intervertebral disc height was measured by lateral plain radiographs. After the radiographic measurements were obtained, the intervertebral discs were removed and analyzed for DNA, sulfated glycosaminoglycan（s-GAG） and water contents of nucleus pulposus. Results： The intervertebral disc height, s-GAG, and water contents in anulus needle punctures were significantly decreased in Group A, but the DNA content in the nucleus pulposus was significantly increased when compared to the control. The significant decrease of disc height and water contents were demonstrated, only the s-GAG and DNA contents did not show a significant difference in Group B when compared to the control. The significant decrease of disc height, s-GAG, water, and DNA contents did not show in Group C when compared to the control. Conclusion： The 18-gauge puncture models produced the most consistent disc degeneration in the rabbit lumbar spine.     

Autologous nucleus pulposus transplantation to lumbar 5 dorsal root ganglion after epineurium discission in rats: a modified model of non-compressive lumbar herniated intervertebral disc

Background  Nucleus pulposus of intervertebral discs has proinflammatory characteristics that play a key role in neuropathic pain in lumbar herniated intervertebral disc. One of the most commonly used animal models (the traditional model) of non-compressive lumbar herniated intervertebral disc is created by L4–L5 hemilaminectomy and the application of autologous nucleus pulposus to cover the left L4 and L5 nerve roots in rats. However, such procedures have the disadvantages of excessive trauma and low success rate. We proposed a modified model of non-compressive lumbar herniated intervertebral disc in which only the left L5 dorsal root ganglion is exposed and transplanted with autologous nucleus pulposus following incision of epineurium. We aimed to compare the modified model with the traditional one with regard to trauma and success rate.

Methods  Thirty Sprague-Dawley male rats were randomized into three groups: sham operation group (n=6), traditional group (n=12), and modified group (n=12). The amount of blood loss and operative time for each group were analyzed. The paw withdrawal threshold of the left hind limb to mechanical stimuli and paw withdrawal latency to heat stimuli were examined from the day before surgery to day 35 after surgery.

Results  Compared with the traditional group, the modified group had shorter operative time, smaller amount of blood loss, and higher success rate (91.7% versus 58.3%, P <0.05). There was no decrease in paw withdrawal latency in any group. The sham operation group had no decrease in postoperative paw withdrawal threshold, whereas the modified and traditional groups had significant reduction in paw withdrawal threshold after surgery (mechanical hyperalgesia).

Conclusions  Transplantation of nucleus pulposus onto the L5 dorsal root ganglion following incision of epineurium in rats established an improved animal model of non-compressive lumbar herniated intervertebral disc with less trauma and more stable pain ethology.

     

BEHAVIOR AND ACTIVITIES IN THE CENTRAL NERVOUS SYSTEM AFTER ACTIVATION OF L-TYPE CALCIUM CHANNELS IN MICE

The dimribmion of the immediave eazly gene c-fos expression in the mouse central nervous system after subcutaneous injection of Bay K 8644 was observed immunohlstochemlcally. Half an after after injection,cfos proecein(FOS) was expzessed in the piriform cortex,sensorimotor cortex,caudate Imtamen,thalamic paraventriculsr nucleus and striate cortex, etc. Intense FOS immunoreactlve (FOS-ir) cells were seen during 2～4 h after injection. The ;results suggested that the distribution of FOS-ir cells aftel subcutaneous injection of Bay K 86LL4 was coincident with that of L-type calcium chentmls ;.rt the different areas of tee CNS. After Bay K 8644 injection, mice appeared seizure-like behavior. The percentage of cells double-labelled by FOS and CaBP immunoreactivities in the observed regions was about 60. 2～72. 8% in CaBP-ir cells. It suggested that most CaBP-ir cells may have L-type calcium channels.     

Techniques for isolating hair cells from guinea pig cochlea.

Isolated live hair cells are important models for studies on the electrophysiology, pathology and pharmacology of the hair cell. Using mechanical isolation method after papain treatment, 70 +/- 27 hair cells, including 0 to 4 inner hair cells, could be obtained from each cochlea of 4 guinea pigs. The criteria for a good viability of isolated cochlear hair cells were: 1) a smooth hair cell membrane; 2) hair cells not swollen; 3) the nucleus in normal position; 4) the cytoplasm in a state of translucence with a halo at the periphery (birefringence); and 5) no Brownian movement of the organelles within the cytoplasm. With short-term culturing at room temperature, approximately 90% of the isolated hair cells retained a good viability at the end of two hours. Subsequently, the hair cells gradually degenerated but still, at the end of five hours, about 40% of them appeared intact. The degeneration patterns have been carefully observed and described.

     

成人脂肪基质细胞体外诱导分化为神经前体细胞的数量达峰时间研究

目的 研究成人脂肪基质细胞(ADSC)诱导分化为神经前体细胞数量达峰时间和诱导分化后神经前体细胞的超微结构特征.方法 应用β-巯基乙醇诱导成人ADSC向神经前体细胞和神经元样细胞分化,倒置相差显微镜观察诱导前后细胞形态;免疫荧光细胞化学染色法检测诱导分化后细胞神经巢蛋白(nestin)的表达情况;透射电镜观察诱导分化3h时的神经前体细胞超微结构.结果 体外培养的成人ADSC诱导分化3h时nestin的表达到高峰,为(86.25±4.82)%,透射电镜观察神经前体细胞的超微结构可见细胞表面突起较多,胞质中细胞器丰富,细胞核大,核/浆比例大,双层核膜且有较多核孔,常染色质多,异染色质少.结论 β-巯基乙醇诱导成人ADSC分化3h时神经前体细胞数量达到高峰,且超微结构研究证实此时细胞处于分裂增殖旺盛期.

Abstract：

Objective To reseach the time point of the highest percentage of neural precursor cells derived from adipose stromal cells (ADSCs) in vitro, and to observe the ultrastructure features of neural precursor cells. Methods Used the β-mercaptoethanol to induce ADSCs to differentiate into neural precursor cells and neuron-like cells. The morphology of the uninductedcells and inducted cells were observed with inverted phase contrast microscope. The expression of nestin which was the marker of neural precursor cell in each group was detected using immunofluorescence staining method. The ultrastructural feature of cells which was induced for 3 hours were observed. Results The highest ratio of positive expression of nestin was 3 hours following induction,with the ratio ( 86.25 ± 4.82) %. There were many protuberance on the cell membrane under transmission electron microscopy.There were plenty of organelles in the neural precursor cells. The neural precursor cells had a large size nucleus,large nucleoplasmic index, much extended chromatin,and less condensed chromatin. The nucleus had double-layer nuclear envelope, more nuclear pore on the nuclear envelope. Conclusion The time point of the highest percentage of neural precursor cells derived from ADSCs is 3 hours,and the ultrastructral feature of induced neural precursor cells confirm that cells at this time point are in a state of split active period.     

Impact of 4HPR on the expression of E-Cad in human bladder transitional epithelial cancer cells T24

Previous researches showed that the expression level of E-Cad in most infiltrating cancer cells was reduced or negative. This study explored whether 4HPR restrained the infiltration of bladder cancer cells through regulating the expression of E-Cad. The infiltrating bladder cancer cells T24 were cultured, and then treated by a proper dosage of drug. Their viability was a determined by MTT method. Western blotting and RT-PCR were adopted to detect the changes of E-Cad gene expression at both pro-tein and mRNA levels. Moreover, immunofluorescent staining and confocal fluorescence microscopy were employed for the observation of the expression of E-Cad. The result showed that, at both mRNA and protein levels, the expression level of E-Cad in T24 cells treated by 4HPR was significantly higher than that of control group, while the β-Cat expression was also relocated from the cell nucleus to cyto-plasm. Our findings suggested that the regulatory function of 4HPR on infiltration of bladder cancer cells T24 is at least partly achieved by regulating the expression of E-Cad.     

Preliminary study on Herpes simplex virus type 1 infection of human oral epithelial cell in vitro

Objective： To explore the functions and mechanisms of herpes simplex virus type I（HSV-1） while infecting human oral epithelial cells in vitro（being similar to the infection in vivo）. Methods：An abundance of HSV-1 strains amplified in Vero cells were used to infect human oral epithelial cells. The culture supernatant was collected to infect Vero cells again. Morphology of HSV-1 was identified by inverted microscope and transmission electron microscope. Nucleic acid of the virus was detected by PCR. Results：The infected human oral epithelial cells didn＇ t display an obvious cytopathic effect（CPE） under inverted microscope（while Vero cells which were infected by the culture supernatant showed typical（CPE）. The virus particles were not observed in the cytoplasm nor in nucleus of human oral epithelial cells, however under transmission electron microscope in the cytoplasm of Vero cells, the nucleic acid of HSV-1 could be detected in infected human oral epithelial cells, by PCR. Conclusion-HSV-1 can successfully infect human oral epithelial cells. This model may provide a useful approach for studying the pathogenesis of herpes virus-associated periodontal disease.     

Extracellular matrix synthesis and ultrastructural changes of degenerative disc cells transfected by Ad/CMV-hTGF-β1

Objective To determine whether the synthesis of proteoglycan, collagen and associated ultrastructure are related to the adenovirus-mediated gene transferred to adult degenerative cells.Methods Adenovirus/cytomegalovirus human transforming growth fector-β1 (Ad/CMV-hTGF-β1) was used to transfect degenerative cells. Antonopulos method, Miamine method and transmission electrion microscopy were conducted to study the synthesis of proteoglycan, collagen, and ultrastructure, respectively. Cell cultures were established from the nucleus pulpous and annulus fibrosus tissues, which were taken from surgery.Results Nucleus pulpous and annulus fibrosus cells were efficiently transduced by the adenoviral vector carrying hTGF-β1 gene. The synthesis of proteoglycan and collagen increased compared with the control group （P&lt;0.05). The metabolism of cells was slightly improved. No significant toxic effects were found.Conclusions Expression of hTGF-β1 gene is efficient to accelerates proteoglycan synthesis and thus accelerates the improvement of collagen. The function and structure of degenerative cells are improved. Ad/CMV-hTGF-β1 may be suitable for treating disc degeneration.     

Effects of Cypermethrin on Male Reproductive System in Adult Rats

Objective To evaluate effects of cypermethrin on the testis histology and testosterone, LH and FSH in adult male Sprague‐Dawley rats. Methods The intact adult male rats were randomly divided into five groups and were treated with cypermethrin at doses of 0, 7.5, 15, 30, or 60 mg/kg per day by oral gavage for 15‐days. After the treatments, serum was collected for hormone assays. The testes, epididymides, seminal vesicles, and prostates were excised and weighed. The right testis was frozen for daily sperm production and the left one was processed for histopathology. Results Daily sperm production decreased significantly in 30 and 60 mg/(kg day) groups. Testicular structure abnormalities included atrophic and distorted seminiferous tubules, deformed and disordered arrangement of germ cells, reduced germ cells, Sertoli cells and Leydig cells, vacuolization and multinucleated formations of spermatids in the cypermethrin‐treated rats. Vacuolization was found in Sertoli cells and the deformed nucleus was noted in Leydig cells. Serum testosterone reduced significantly in 30 and 60 mg/(kg day) groups. Serum FSH increased significantly in 60 mg/(kg day) group. Conclusion Cypermethrin induces impairments of the seminiferous tubules structure and spermatogenesis in the rats. The damages of the male reproductive system may be attributed to the imbalance of circulating testosterone.