肿瘤免疫治疗很早就被提出,也成为继肿瘤传统治疗方法 (化学治疗、手术治疗及放射治疗)之后的第四种肿瘤治疗方式.这种方法 的出现曾给肿瘤患者带来希望,但免疫治疗的进展并不突出.近几年,随着肿瘤学与免疫学的发展,免疫治疗逐渐发挥更加重要的作用,同时免疫学也拓展了人们对肿瘤的新视野,使得以前以肿瘤细胞为中心的观点发展为对肿瘤生物学行为复杂的、整体的认知.肿瘤免疫治疗方法 主要有以T细胞为主的治疗、肿瘤疫苗治疗及以抗体为主的治疗.本文主要介绍肿瘤免疫治疗新进展以及面临的挑战.

PURPOSE:To compare the effects of 25 kDa full-length rhAm and porcine EMPs on cell behaviors of human periodontal ligament fibroblasts (HPDLF) and foreskin fibroblasts(HFF). METHODS:rhAm was induced by BL21/pET28a-His-SUMO-rhAm express system, and 25 kDa full-length rhAm was analyzed by SDS-PAGE and Western blot. EMPs were extracted by acetic acid method. HPDLF and HFF were cultured in vitro. The cells were treated with rhAm and EMPs at different concentrations. The cell adhesion, proliferation and migration assays were qualitatively analyzed. The data was statistically analyzed with SAS 5.0 software package. RESULTS:10-20 μg/mL rhAm significantly promoted the adhesion, proliferation and migration of HPDLF and HFF (P<0.05), but no significant difference between two proteins was found (P>0.05). CONCLUSIONS:25 kDa rhAm and EMPs shows similar biological effects on fibroblast, which indicates that rhAm may play an important role in the periodontal regeneration through the activation of fibroblasts.

Objective To evaluate the behavior of bone marrow mesenchymal stem cells(BMMSCs) on novel SrCa_2ZnSi_2O_7,(Sr-HT) coating in vitro.Methods The SEM experiment was performed to characterize the morphology of SrHT coating.The accumulated ion concentrations of the dissolution products were measured using an inductively coupled plasma/optical emission spectroscopy(ICP-OES).Ca_2ZnSi_2O_7(HT) and hydroxyapatite(Hap) coatings were used as control groups and the adhesion and osteogenic differentiation of BMMSCs on the surfaces of different coating were evaluated and analyzed.Results Sr-HT,HT and HAp coatings had a microscale structure at low magnification and their main difference was ion composition of extracts.Compared to HAp coating,a large number of Zn ions were detected in the extracts of the Sr-HT and HT coatings and plenty of Sr ions were released from Sr-HT coating.The adhesion and osteogenic differentiation of BMMSCs cultured on Sr-HT and HT coatings were significantly higher than that on HAp coating.Besides,Sr-HT coating displayed the best osteogenic differentiation effect on BMMSCs.Conclusions The novel Sr-HT coating has a positive effect on BMMSCs adhesion and osteogenic differentiation as compared with the traditional HAp coating.

Objective: The aim of this study was to examine the osteogenic differentiation potential of dental pulp stem cells from goat deciduous teeth(SGDs) in vitro.Methods: SGDs were grown in modified tissue culture medium and cells from the third passage were used for experimentation.SGDs with porous calcium phosphate cement(pCPC) were transplanted into the goat left dorsal muscle pouch,and the pCPC without cells were transplanted into the right dorsal muscle pouch as negative controls.The transplants were harvested at the end of 4,8 weeks and prepared for histological evaluation.Results: Examination showed obvious bone formation at 4 weeks in SGDs-pCPC group and the amount of bone formation increased at 8 weeks after transplantation.Bone formation was not detected in only pCPC group.Conclusion: SGDs express the potential to differentiate into osteoblasts in vitro;SGDs combined with scaffold such as pCPC have the ability of ectopic osteogenesis.

The aim of this study was to explore the effects of maxillary sinus floor elevation and simultaneous dental implantation with a tissue-engineered bone complex of calcium phosphate cement (CPC) scaffolds combined with bone marrow stromal cells (BMSCs). A large animal goat model is used with the tissue engineering method. Eighteen bilateral maxillary sinus of nine goats were randomly allocated into three groups; the CPC/BMSC complex (n=6) was used to elevate maxillary sinus floor with a simultaneous implant placement; the effects were compared with those treated with CPC alone (n=6) or autogenous bone (n=6). After a healing period of 3 months, sequential triad-color fluorescence labeling, micro-CT, as well as histological and histomorphometric analyses indicated that the tissue-engineered BMSC/CPC complex could promote earlier bone formation and mineralization, and maximally maintain the volume and height of the augmented maxillary sinus. By comparison, CPC-alone or autogenous bone achieved less bone formation and later mineralization. Besides, the average bone-implant contact value reflecting the osseointegration was 35.63%±9.42% in the BMSCs/CPC group, significantly higher than 22.47%±4.28% in the CPC-alone group or 28.26%±8.03% in the autogenous bone group. In conclusion, CPC serves as a potential substrate for BMSCs for the maxillary sinus floor augmentation and simultaneous implantation. The tissue-engineered bone might enhance the stability of implants and thus be of great significance to achieve improved quality to restore the oral function in clinic.

PURPOSETo investigate ectopic osteogenesis of composites SGDs with porous calcium phosphate cement(pCPC) in goat's muscle pouch.METHODSSGDs were cultivated with modified tissue culture techniques, then were induced into osteoblasts in the third passage, the osteogenic-induced SGDs were combined with pCPC and transplanted into the goat left dorsal muscle pouch, the pCPC without cells was transplanted into the right dorsal muscle pouch as negative controls. The transplants were harvested at 2-,4-,6-,8- week and prepared for histological examination. The morphologic quantitative analysis was made by SPSS 16.0 software package.RESULTSBone formation was not detected in pCPC without cells by histological examination. 2,4,6,8 weeks after transplantation in SGDs-pCPC group, the percentages of bone formation were (1.24±0.25)%,(1.59±0.23)%,(4.12±0.39)% and (5.68±0.58)%,respectively.There was no significant difference in bone formation at 2 and 4 weeks after transplantation (P>0.05). At 8- week, the percentages of bone formation were higher than that at 6- week in SGDs-pCPC group, and both significantly higher than that at 2- and 4- week(P<0.05).CONCLUSIONSGDs combined with pCPC have the ability of ectopic osteogenesis.

PURPOSE:To observe the biocompatibility and ectopic bone-like tissue formation of stem cells from goat deciduous teeth (SGDs) with porous calcium phosphate cement (pCPC).METHODS:The expression of STRO-1 on SGDs was measured with flow cytometry (FCM); the 4th passage SGDs were cultured in induced-mineralization medium in vitro for 7 days. Combined with pCPC, the adhesion and growth of the compounds were observed with scanning electron microscopy (SEM); the ectopic bone-like tissue formation was observed 8 weeks after the compounds implanted subcutaneously into the nude mice.RESULTS:On the third day of SGDs compounded with pCPC, SEM verified that the cells adhered closely and tightly with pCPC, protruded pseudopods and secreted matrix. 8 weeks after the compounds implanted in ectopic sites, HE staining confirmed the formation of bone-like tissue; Immunohistochemistry showed the strongly positive expression of OCN protein in the implanted materials.CONCLUSIONS:SGDs may differentiate into osteoblast and are potential to induce bone matrix formation; combined with pCPC, the compounds may generate bone-like tissue.

To explore the possible mechanism of osteogenesis for deciduous teeth stem cells (DTSCs) in vivo/ vitro, stem cells from goat deciduous teeth (SGDs) were firstly isolated, induced and transplanted into immunocompromised mice. The SGDs’s mineralization pattern and osteogenesis were compared with bone marrow messenchymal stem cells (BMMSCs) from goats. SGDs have similar osteogenic differentiation pattern in vitro and bone-like tissue formation mechanism in vivo to BMMSCs; moreover SGDs have stronger alkaline phosphatase (ALP) gene expression and osteopontin (OPN) gene expression levels than BMMSCs; also SGDs can form more bone-like tissues than BMMSCs when cell-scaffold compounds are transplanted into immunocompromised mice. This pre-clinical study in a large-animal model confirms that DTSCs may be an appropriate source of stem cells in repairing bone defects with tissue engineering.

Objective To construct the recombinant eukaryon expression plasmid containing human amelogenin(hAm) gene and transfect mammalian cell line NIH3T3 for construction of cells with stable expression of recombinant hAm.Methods hAm gene was inserted into eukaryon expression vector pcDNA3.1/myc-His(-) A with restriction enzyme EcoRⅠ and BamHⅠ,and recombinant plasmid pcDNA3.1/myc-His(-) A-hAm containing hAm gene was confirmed by restriction endonuclease mapping and sequencing.pcDNA3.1/myc-His(-) A-hAm was transfected into NIH3T3 cells by LipofectamineTM2000,and was selected by G418 for positive cell clones.Cells with stable expression of hAm was constructed,and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting. Results Restriction endonuclease mapping and sequencing revealed that the inserted sequences were accurate in recombinant plasmid pcDNA3.1/myc-His(-)A-hAm.Expression of hAm with molecular weight of 28 000 was detected by SDS-PAGE and Western blotting in NIH3TS cells transfected with recombinant plasmid,which was in line with the prediction. Conclusion The recombinant eukaryon expression system containing hAm has been successfully constructed,and NIH3T3 cells with stable expression of recombinant hAm is obtained.

PURPOSE:To isolate and cultivate dental cells from goat deciduous teeth ,and explore changes of its biological characters before and after induced-mineralization.METHODS:Pulp cells were cultivated with modified tissue block enzymolytic method, cell lineage in the second passage with SAB methods was checked out. Induced-mineralized cultivation was adopted in the fourth passage, some examinations were used to compare with normal cultivated cells: cell proliferative capality, mineralized ability test, cell morphology change, protein(OCN) expression level, related osteogenic genes(ALP,COL-I,OCN,OPN) expression.RESULTS:Modified tissue block enzymolytic method could culture better pulp cells derived from goat deciduous teeth. Immunohistochemical staining showed that pulp cells were from mesenchyma. MTT method showed that induced-mineralization pulp cells proliferated more slowly than un-induced cells. Compared with uninduced-mineralization cells, induced-mineralization cells had stronger ALP activity and Alizarin red staining rate, its proteins(OCN) and mineralized genes(ALP,OCN) expression were significantly upregulated.CONCLUSIONS:Pulp cells can be cultivated derived from goat exfoliated deciduous teeth with modified tissue block enzymolytic method. Fourteen days after continuous induced-mineralization culture , pulp cells derived from the goat deciduous teeth might own the potential in differentiating to osteoblast and form bone-like tissue.

PURPOSE:To isolate and culture pulp cells from goat deciduous teeth, and transfect green fluorescent protein gene.METHODS:Pulp cells from goat deciduous teeth were obtained by tissue culture. Cell growth curve was measured by counting the number of cells, HE and alkaline phophatase(AKP) stain, as well as immunhistochemical stain of vimentin and keratin were performed. Virus supernatant was used to infect cell green fluorescent protein gene.RESULTS:For the pulp cells, the cell group double time was 43.79 hours. AKP stain and immunhistochemical stain of vimentin were both positive, while immunhistochemical stain of keratin was negative. The infected cells expressed green fluorescent.CONCLUSIONS:Pulp cells could be cultured from goat deciduous teeth, and express green fluorescent successfully.

目的:观察氯化镧(LaCl2)干预后的体外培养的骨髓基质细胞(BMSCs)与脱钙冻干骨(MDBM)复合后的异位威骨能力.方法:将经过5.564×102、5.554、5.564×10-2μg/mL 3种浓度La3+干预的第3代BMSCs、空白对照组和骨形成蛋白-2阳性对照组细胞与fdDBM复合后回植入裸鼠皮下,8周后对回植标本做组织学分析、X线密度测定及钙磷含量测定.结果:回植物组织切片显示各组标本均可见新生的骨组织样结构.La3+干预各组的X线密度及钙磷含量均数与空白支架组比较差异均有显著性.但La3+"干预各组组间差异不具统计学意义.结论:5.564×102、5.564、5.564×102μg/mL 3种浓度的La3+均能促进组织工程骨矿化,以5.564 μg/mL的LaCl3促进骨矿化作用较强.

PURPOSE:To evaluate the biocompatibility of oxide film formed by micro-arc oxidation(MAO) on titanium surface by culturing bone marrow stromal cells(BMSCs) on its surface and observing the BMSCs's biological behavior of early adhesion, growth and alkaline phosphatase activity.METHODS:According to the surface treatments, the samples were classified into 4 groups: MAO group, abrator group, abrator and NaOH-treated group, and untreated titanium group. Attachment, growth and alkaline phosphatase (ALP) activity of BMSCs cultured on the titanium surface of each group were assessed at different time. All data were statistically analyzed with one-way ANOVA using SPSS11.5 software package.RESULTS:From a morphological point of view, cell adherence in the MAO group was the best. Cell count and ALP activity in the MAO group were highest(P<0.01).CONCLUSIONS:MAO film on pure titanium surface may increase BMSCs adhesion, growth and ALP activity and titanium surface treated with MAO exhibits better biocompatibility.

PURPOSE: To examine the ability of Nell-1 (Nel-like, type 1 molecule) gene in inducing osteogenic differentiation of bMSCs in vitro. METHODS: bMSCs obtained from Fischer 344 rats were transferred with Ad-Nell-1 or Ad-β-Galactosidase (Ad-LacZ) or left untransferred in vitro. Expression of Nell-1 gene was detected by reverse transcription-PCR (Rt-PCR) and Western blot. Alkaline phosphatase (ALP) activity and osteocalcin (OC) production were measured and Von Kossa test was conducted to examine Nell-1’s ability in inducing osteogenic differentiation of bMSCs. The data were analyzed for ANOVA with SNK method using SAS 6.04 software package (P0.05). RESULTS: Nell-1 transgenic message and protein were detected in Nell-1 group but not in LacZ group and untransferred group. On day 3,6, 9 after gene transfer, ALP activity was significantly enhanced in the Nell-1 group. On day 14,21 after gene transfer, OC production in Nell-1 group was (1.23±0.05)ng/mL and (1.31±0.06)ng/mL, significantly higher than those in untransferred group(0.81±0.09)ng/mL and (1.00±0.05)ng/mL and LacZ group(0.92±0.08)ng/mL and (1.02±0.04)ng/mL].On day 21,28 after gene transfer, the nodules formed in Nell-1 group was 4.5±1.1 and 16.2±2.5 significantly, higher than those in untransferred group (0.8±0.7 and 3.2±1.2) and LacZ group(0.7±0.5 and 3.7±0.8). CONCLUSION: Nell-1 gene induce osteogenic differentiation of bMSCs in vitro.

Objective To prepare seed cells for bone tissue engineering,establish the culturing condition and biological characteristics of canine bone marrow stromal cells(BMSCs),and proliferate them into osteoblasts in vitro.Methods Bone marrow was drew out from Beagle dogs and cultured with differentiation culture medium to harvest BMSCs.The morphology of the cells was studied with a phase contrast microscope.Von Kossa staining and alkaline phosphatase(ALP) activity test were employed to assess BMSCs osteoblastic differentiation and the generation of calcified extracellular matrix.The standard curves of cell-amount vs OD-value were drawn through spectrophotometer test and MTT assay.Results BMSCs could be observed after 18 days of primary culture.The viable characteristics of BMSCs were relatively stable,and these cells possessed bone formation potentials.Conclusion BMSCs can be cultured,differentiated and proliferated with active osteogenic function with differentiation culture medium in vitro,thus they can be candidate cells for bone tissue engineering.

PURPOSE To detect the genotoxicity of dental machinable ZrO(2)/LaPO(4) diphase ceramics on human peripheral blood lymphocytes in vitro. METHODS The evaluation of DNA damage on human lymphocytes was performed by comet assay for three groups of ZrO(2)/LaPO(4) diphase ceramics with 30wt% of LaPO(4) (with 3wt% and 5wt% of Y(2)O(3)) and 40wt% of LaPO(4) (with 5wt% of Y(2)O(3)). The results were analyzed with SPSS16.0 software package for one-factor ANOVA and LSD. RESULTS Three experimental groups with different concentration of LaPO(4) of ZrO(2)/LaPO(4) diphase ceramics, the negative control of IPS Empress II ceramics and the blank behaved little migration of the DNA strands respectively after six-day test, and there was no significant difference in all the groups except the positive control (P>0.05). CONCLUSION The study indicates little effect of DNA damage of ZrO(2)/LaPO(4) diphase ceramics.

PURPOSETo investigate the effect of hypoxia on proliferation and expression of HIF-1alpha and Caspase-3 in human periodontal ligament cells (PDLCs).METHODSHuman PDLCs were exposed to cobalt chloride in order to mimic hypoxia. Cell viability of PDLCs was determined by MTT methods. Expression of HIF-1alpha and Caspase-3 was measured by real time PCR and Western blot. The data was statistically analyzed with SAS6.12 software package for one-way ANOVA.RESULTSCell viability of PDLCs significantly decreased when exposed to hypoxia in a time- and dose-dependent manner. Hypoxia induced the expression of HIF-1alpha,up-regulated the expression of Caspase-3.CONCLUSIONSHypoxia inhibits cell proliferation, which involves the expression of HIF-1alpha and Caspase-3, resulting in the production of the apoptosis. The results suggest that hypoxia may play a role in the induction and progression of chronic periodontitis. Supported by National Natural Science Foundation of China(Grant No.30801292), Shanghai Leading Academic Discipline (Grant No.S30206) and Research Fund for Excellent Young Teachers of Shanghai Municipal College and University (Grant No.JDY07059).

PURPOSE:To study the bone differentiation related gene expression ALP,OCN,COL I changes in mRNA level after goat bone marrow mesenchymal stem cells(BMSCs) inducing to osteoblast.METHODS: Whole bone marrow culture method was used to amplify the BMSCs in vitro.Bone formation conditioned medium was adopted to induce the BMSCs differentiate to osteoblast.Characterization was performed by using cell morphology observation,immuno-histochemical staining and Von Kossa methods.RT-PCR and real-time PCR were used to examine the bone differentiation related gene OCN,collagen I,ALP mRNA expression.The untreated BMSCs were used as the control.RESULTS: After osteoblast characterization,the BMSCs were successfully differentiated to osteoblast.The OCN,collagen I mRNA expression were up-graded,while ALP mRNA expression had little change.CONCLUSION: BMSCs can be successfully differentiated to osteoblast by using bone formation conditioned medium.The bone differentiation related genes change to accommodate the osteoblast function.

PURPOSE:To construct the recombinant lentiviral vector of human amelogenin gene, infect human periodontal ligament cells with the recombinant lentivirus, and evaluate the feasibility of applying modified PDLCs as seeds for a further periodontal reconstruction.METHODS:The mature peptide of hAm cDNA was cloned and linked into the vector plasmid, the recombinant plasmid FUAmW was confirmed by double enzyme digestion and sequence analysis. Recombinant lentivirus was prepared from 293T cells by polytheylenimine (PEI)-mediated transient cotransfection. The hPDLCs and 293T cells were infected with the generated lentivirus. The infection efficiency was analysed by detection of green fluorescence protein (GFP) with fluorescent microscope and flow cytometer 72 hours later. The expression of hAm gene was detected by reverse transcription polymerase chain reaction (RT-PCR).RESULTS:The sequence of inserted fragment in recombinant plasmid was identical to the hAm sequence reported in Genebank. Green fluorescence was visible under fluorescent microscope, FCM assay showed that positive percentage was 69.46% and 33.99% in 293T and hPDLCs, respectively. The targeted gene was obtained in the experimental groups by RT-PCR.CONCLUSIONS:The recombinan lentiviral vector of hAm gene is constructed successfully and it could be transfected into cultured hPDLCs. hAm gene and seed cells may be used for further study in the fields periodontal tissue engineering. Supported by National Natural Science Foundation of China (Grant No. 30672315).

AIM:To investigate the expression and regulation of matrix metalloproteinase in adult odontobast.METHODS:The expression of MMP-2,-9 and-8 in odontoblast and the effect of TGF-β1 on its secretion were measured using enzymography,Western blot and immunohistochemical staining.RESULTS:With enzymography and Western blot,MMP-2,-9 and-8 expressed in odontoblast,and MMP-2 and-8 were upregulated and MMP-9 was downregulated by TGF-β1.Immunohistochemical staining confirmed that MMP-8 expressed in odontoblast layer.CONCLUSION:The adult odontoblast is one of the source of MMP-2,-9 and-8,and TGF-β1 may regalate their expression.