Progranulin promotes osteogenic differentiation of human periodontal ligament stem cells via tumor necrosis factor receptors to inhibit TNF-α sensitized NF-kB and activate ERK/JNK signaling.

OBJECTIVE: To investigate the molecular mechanism of Progranulin (PGRN) in promoting osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in inflammatory environment. BACKGROUND: Progranulin is an antagonist of tumor necrosis factor (TNF) receptors (TNFRs) and is known to promote inflammatory periodontal bone defect regeneration. METHODS: TNFR1- and TNFR2-silenced hPDLSCs designed as hPDLSCs-sh-TNFR1 and hPDLSCs-sh-TNFR2 were cultured with osteoinductive medium containing TNF-α and (or) PGRN. Immunofluorescence, quantitative real-time PCR, and western blot were used to, respectively, detect expressions of TNFR1\TNFR2 and osteogenic differentiation markers as well as phosphorylation level in NF-κB\MAPK-related pathways. RESULTS: Immunofluorescence and real-time PCR showed that TNFR1 and TNFR2 positively expressed in hPDLSCs. TNF-α stimulation could significantly decrease the expressions of ALP and RUNX2 in hPDLSCs, whereas PGRN treatment could significantly enhance their expressions, and reverse TNF-α-mediated expression suppression of ALP and RUNX2 in hPDLSCs. In hPDLSCs-sh-TNFR1, TNF-α mediated osteogenic inhibition decreased, but both TNF-α + PGRN and alone PGRN significantly promoted expression of ALP and RUNX2. PGRN significantly enhanced expression of P-ERK1/2 and P-JNK, while corresponding inhibitors eliminated PGRN-stimulated osteogenic differentiation. In hPDLSCs-sh-TNFR2, no significant difference existed in osteogenic markers and P-JNK expression between the PGRN group and the control group. However, PGRN still activated P-ERK1/2 expression. Besides, PGRN antagonized TNF-α-enhanced NF-κB P65 expression. CONCLUSION: Progranulin promotes osteogenic differentiation of hPDLSCs via TNFR1 to inhibit TNF-α-sensitized NF-κB and via TNFR2 to activate JNK signaling. The mechanism by which PGRN activates ERK signaling remains to be explored.

Integrated analysis of lncRNA-miRNA-mRNA ceRNA network in squamous cell carcinoma of tongue.

BACKGROUND: Numerous studies have highlighted that long non-coding RNAs (lncRNAs) can bind to microRNA (miRNA) sites as competing endogenous RNAs (ceRNAs), thereby affecting and regulating the expression of mRNAs and target genes. These lncRNA-associated ceRNAs have been theorized to play a significant role in cancer initiation and progression. However, the roles and functions of the lncRNA-miRNA-mRNA ceRNA network in squamous cell carcinoma of the tongue (SCCT) are still unclear. METHODS: The miRNA, mRNA and lncRNA expression profiles from 138 patients with SCCT were downloaded from The Cancer Genome Atlas database. We identified the differential expression of miRNAs, mRNAs, and lncRNAs using the limma package of R software. We used the clusterProfiler package for GO and KEGG pathway annotations. The survival package was used to estimate survival analysis according to the Kaplan-Meier curve. Finally, the GDCRNATools package was used to construct the lncRNA-miRNA-mRNA ceRNA network. RESULTS: In total, 1943 SCCT-specific mRNAs, 107 lncRNAs and 100 miRNAs were explored. Ten mRNAs (CSRP2, CKS2, ADGRG6, MB21D1, GMNN, RIPOR3, RAD51, PCLAF, ORC1, NAGS), 9 lncRNAs (LINC02560, HOXC13 - AS, FOXD2 - AS1, AC105277.1, AC099850.3, STARD4 - AS1, SLC16A1 - AS1, MIR503HG, MIR100HG) and 8 miRNAs (miR - 654, miR - 503, miR - 450a, miR - 379, miR - 369, miR - 190a, miR - 101, and let-7c) were found to be significantly associated with overall survival (log-rank p < 0.05). Based on the analysis of the lncRNA-miRNA-mRNA ceRNA network, one differentially expressed (DE) lncRNA, five DEmiRNAs, and three DEmRNAs were demonstrated to be related to the pathogenesis of SCCT. CONCLUSIONS: In this study, we described the gene regulation by the lncRNA-miRNA-mRNA ceRNA network in the progression of SCCT. We propose a new lncRNA-associated ceRNA that could help in the diagnosis and treatment of SCCT.

Anticancer Effects of Emodin on HepG2 Cell: Evidence from Bioinformatic Analysis.

Hepatocellular carcinoma (HCC) is a primary cause of cancer-related death in the world. Despite the fact that there are many methods to treat HCC, the 5-year survival rate of HCC is still at a low level. Emodin can inhibit the growth of HCC cells in vitro and in vivo. However, the gene regulation of emodin in HCC has not been well studied. In our research, RNA sequencing technology was used to identify the differentially expressed genes (DEGs) in HepG2 cells induced by emodin. A total of 859 DEGs were identified, including 712 downregulated genes and 147 upregulated genes in HepG2 cells treated with emodin. We used DAVID for function and pathway enrichment analysis. The protein-protein interaction (PPI) network was constructed using STRING, and Cytoscape was used for module analysis. The enriched functions and pathways of the DEGs include positive regulation of apoptotic process, structural molecule activity and lipopolysaccharide binding, protein digestion and absorption, ECM-receptor interaction, complement and coagulation cascades, and MAPK signaling pathway. 25 hub genes were identified and pathway analysis revealed that these genes were mainly enriched in neuropeptide signaling pathway, inflammatory response, and positive regulation of cytosolic calcium ion concentration. Survival analysis showed that LPAR6, C5, SSTR5, GPR68, and P2RY4 may be involved in the molecular mechanisms of emodin therapy for HCC. A quantitative real-time PCR (qRT-PCR) assay showed that the mRNA levels of LPAR6, C5, SSTR5, GPR68, and P2RY4 were significantly decreased in HepG2 cells treated with emodin. In conclusion, the identified DEGs and hub genes in the present study provide new clues for further researches on the molecular mechanisms of emodin.

[Construction and identification of cbfa1 and satb2 co-expression vector].

PURPOSE: To construct a lentiviral eukaryotic expression vector containing two genes of cbfa1 and satb2. METHODS: The aim genes of cbfa1 and satb2 were amplified from plasmids by PCR. After TA cloning, the positive clones were identified by restrictive enzyme digestion and commercial DNA sequencing. Cbfa1 and satb2 with correct sequences were ligated upstream and downstream to pIRES, respectively, to construct pIRES-cbfa1-satb2. Then cbfa1-Ires-satb2 fragment was obtained by double digestion, and inserted into corresponding enzyme cut sites of pLentinTrident1-CMV which had been added resistance gene neo/kana to construct the lentiviral eukaryotic expression vector pLentinTrident1-CMV-cbfa1-Ires-satb2. RESULTS: We amplified the genes cbfa1 and satb2 by PCR and connected them with pLentinTrident1-CMV by internal ribosomal entry site of mediator pIRES successfully. The result was identified by PCR, restrictive enzyme digestion and sequencing. CONCLUSIONS: Recombinant lentiviral eukaryotic expression vector containing both cbfa1 and satb2 genes is successfully constructed. This provides a foundation for further studies on their functions.

[Quantitative detection of human cytomegalovirus in aggressive and chronic periodontitis lesions].

OBJECTIVE: To establish a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA load in subgingival specimens from the patients with aggressive and chronic periodontitis, and to investigate the relationship between HCMV infection and the periodontal status. METHODS: A total of 114 subgingival plaque specimens were taken from 18 subjects with aggressive priodontiti (AgP), 24 subjects with chronic periodontitis (CP) and 15 healthy control subjects. Standard quantification was performed with recombinant plasmid containing a conserved fragment of HCMV. The SYBR Green I fluorescent quantitative real-time PCR assay was established based on positive plasmid. HCMV DNA load in the specimens were detected with quantitative real-time PCR based on SYBR Green I fluorescence. RESULTS: HCMV were detected in 58.3% of AgP sites and 41.7% of CP sites, however, only 6.7% of periodontally-healthy sites were HCMV positive. The detection rate of HCMV in periodontitis lesions was significantly higher than in periodontal health (P < 0.01). High copy-counts more than 10(4) of HCMV were detected in 33.3% of AgP sites, which were significantly higher than in CP sites (10.4%) (P < 0.05). CONCLUSION: Subgingival infection with HCMV is closely associated with periodontitis. Active HCMV infection may be related to the rapid tissue destruction of AgP.

[Effects of high mobility group box 1 in activating periodontal ligament fibroblasts to express cytokine].

OBJECTIVE: To investigate the influence of high mobility group box 1 (HMGB1) on the expression of interleukin 6 (IL-6), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) on periodontal ligament fibroblasts. METHODS: Human periodontal ligament fibroblasts were stimulated with HMGB1 at concentrations of 10, 30, and 100 ng x mL(-1) for 24 h. RT-PCR and Western blot analysis were performed to check mRNA and protein expression of IL-6, RANKL and OPG on the cells. RESULTS: The ratio of RANKL/OPG was increased at both mRNA and protein level after HMGB1 stimulation at 10, 30, 100 ng x mL(-1). Inflammatory cytokine IL-6 was upregulated by HMGB1 at the concentration of 100 ng x mL(-1). CONCLUSION: Increased ratio of RANKL/OPG and IL-6 on periodontal ligament fibroblasts suggests that HMGB1 might play a role in the pathogenesis and progression of periodontal disease.

[Levels of inflammation cytokines in patients with coronary heart disease and periodontal disease].

OBJECTIVE: To determine the level of high-density lipoprotein cholesterol (HDL-C), serum C-reactive protein (CRP) and inflammation cytokines and investigate the concentration between periodontal disease and coronary heart disease (CHD). METHODS: Sixty-six patients with CHD and chronic periodontitis [(C+P) group], forty-four with only CHD (C group), fifty-six with only chronic periodontitis (C group), and forty-three healthy controls (H group) were included in this study. The diagnosis of chronic periodontitis and CHD was based on accepted clinical criteria. Serum levels of HDL-C, CRP, IL-6, tumor necrosis factor (TNF)-alpha and IL-1beta were tested in all patients and controls. RESULTS: The periodontal conditions of these four groups were significantly different (P<0.05). The clinical periodontal parameters [probing depth (PD), attachment loss (AL), and bleeding on probing (BOP)] in patients with (C+P) group, P group, C group and H group were [(4.55+/-0.85) mm, (3.78+/-0.34) mm, 69.6%], [(4.06+/-0.61) mm, (3.05+/-0.44) mm, 63.6%], [(1.85+/-0.67) mm, (1.26+/-0.39) mm, 20.5%], [(1.12+/-0.33) mm, (0.42+/-0.83) mm, 4.6%], respectively. The levels of HDL-C in H group, C group, P group and (C+P) group were (1.42+/-0.21), (1.22+/-0.18), (1.24+/-0.21) and (1.04+/-0.22) mmol/L, respectively. T compare with other three groups, the level of HDL-C in (C+P) group is the lowest. The levels of CRP, IL-6, TNF-alpha and IL-1beta in (C+P) group were significantly higher than other groups (P<0.05). CONCLUSIONS: HDL-C, CRP, IL-6, TNF-alpha and IL-1beta may be associated with the pathogenesis of periodontitis and CHD. There may be a relationship between the two diseases.

[Detection of platelet-activating factor in unstimulated mixed saliva and gingival crevicular fluid of periodontitis patients with coronary heart disease].

PURPOSE: To explore the correlation between periodontitis and coronary heart diseases(CHD) by comparing the levels of platelet-activating factor (PAF) in saliva and gingival crevicular fluid (GCF) among 4 groups. METHODS: One hundred and twenty subjects were divided into 4 groups according to the diagnostic angiography and periodontal examination as group (C+P), group C, group P and group H.Group (C+P) was made up of 24 CHD patients with moderate to advanced periodontitis.36 CHD patients without periodontitis were as group C.Group P was made up of 32 patients suffering moderate to advanced periodontitis.Group H was 28 healthy volunteers. The general information and periodontal status of all the subjects were recorded and unstimulated mixed saliva and GCF samples were collected.The PAF level was detected by ELISA. Then the difference in PAF level of each group was analyzed with SPSS13.0 software package. RESULTS: One way analysis of variance showed that the levels of PAF in saliva and GCF from the patients of group (C+P) and P were significantly higher than the group C and group H. The results of Pearson correlation analysis showed that the levels of PAF in saliva and GCF positively correlated with pocket depth (PD) and attachment loss (AL) (P<0.05). CONCLUSIONS: Inflammation of periodontal tissues can increase the level of PAF which may be a risk factor of coronary heart diseases. It indicates that the raising level of PAF may be another significant factor in the process of periodontitis affecting the progress of coronary heart disease.

A pilot study evaluating the effect of recombinant human bone morphogenetic protein-2 and recombinant human beta-nerve growth factor on the healing of Class III furcation defects in dogs.

BACKGROUND: The quantity of regenerated bone induced by recombinant human bone morphogenetic protein-2 (rhBMP2) is encouraging, but sometimes the quality is inferior. Recombinant human beta-nerve growth factor (rh beta-NGF) plays a major role in bone remodeling. This study evaluates the quality and quantity of regenerated bone in periodontal regeneration following topical application of the two growth factors to Class III furcation defects. METHODS: Thirty-six inflamed Class III furcation defects were created in six beagle dogs at sites of mandibular premolars 2, 3, and 4, and then biodegradable hydrogel incorporating rhBMP2 and rh beta-NGF was topically applied to the defects. The groupings were as follows: G1, untreated (control group A); G2, carrier alone (control group B); G3, 0.4% rhBMP2 + carrier; G4, 2% rh beta-NGF + carrier; G5, 0.4% rhBMP2 + 2% rh beta-NGF + carrier; and G6, 0.2% rhBMP2 + 1% rh beta-NGF + carrier. Eight weeks after application, the quality and quantity of regenerated tissue were evaluated by scanning electron microscopy observation, calcium/phosphorus ratio analysis, and histologic evaluation. RESULTS: The regenerated bone in G5 exhibited the highest calcium/phosphorus ratio among all groups and showed a denser structure with more calcified substances on the collagen fiber surface than that in the other groups. Histomorphometric analysis revealed that 0.4% rhBMP2 + 2% rh beta-NGF promoted the highest percentage of periodontal regeneration among all groups. CONCLUSION: The results of this pilot study suggest that a topical application of rhBMP2 and rh beta-NGF may improve the quality and quantity of regenerated bone in artificially created Class III furcation defects of beagle dogs.

[Research advances in the mechanism of phenytoin-induced gingival overgrowth].

Phenytoin is widely used as an antiepileptic drug in clinic, but a common and well-known adverse effect of its administration is gingival overgrowth which may influence the aesthetics and function of the oral cavity. The pathogenesis of phenytoin-induced gingival overgrowth is not very clear now. This review summarized the influences of phenytoin on gingival fibroblast,collagen homeostasis and some cytokines, etc.

[Ultrasound-mediated microbubble destruction enhances bone morphogenetic protein-2 gene expression in mouse skeletal muscles].

OBJECTIVE: To explore whether ultrasound-mediated microbubble destruction can enhance the expression efficiency of plasmid pIRES-rhBMP2-EGFP for bone morphogenetic protein-2(BMP-2) in mice skeletal muscle. METHODS: Twenty four male BALB/c mice were divided into four groups. The naked plasmid was injected into the pretibial muscle or the quadriceps muscle (group A and group C) without ultrasound-mediated microbubble destruction method. Micobubbles with plasmid were injected into the pretibial muscle or the quadriceps muscle (group B and group D) with destructing microbubbles by ultrasound immediately. Twelve mice (group A and group B, 30 microg plasmid injected) were killed after 7 days and the tissue samples of the pretibial muscle were obtained to observe the expression of enhanced green fluorescence protein (EGFP) by inverted fluorescence microscope, gene transfection efficiencies were quantified by counting EGFP positive fibers on mice skeletal muscle. After 14 days, the other twelve mice (group C and group D, 100 microg plasmid injected) were killed and immunnohistochemical technique was applied to detect the rhBMP-2 gene expression. RESULTS: The percentage of GFP-positive fibers was significantly lower in the group A than that in the group B. After 14 days, expression of rhBMP-2 was detected in cells and interstitial spaces in the group C and group D, and expression efficiency of rhBMP-2 in the group D was significantly higher than that in the group C. CONCLUSION: Ultrasound-mediated microbubble destruction could enhance the transfection and expression efficiency of rhBMP-2 gene in skeletal muscle of mouse in vivo. It is a new gene therapy method for periodontal regeneration.

[Ultrasound-mediated microbubble destruction enhances exogenous gene expression in NIH3T3 cells in vitro].

OBJECTIVE: To investigate the transfection efficiency of the recombinant human bone morphogenetic protein-2 (hBMP-2) gene in targeted cells by ultrasound-mediated microbubble destruction. METHODS: NIH3T3 cells' anabiosis was completed and went down to the 3rd or 4th generation, and cultured in 6 well plates. The cells were divided into 2 groups: Plasmid DNA and Lipofectamine 2000 group (liposome group), plasmid DNA and ultrasound and microbubble group (ultrasound-mediated microbubble destruction group). Plasmid DNA was transfected into cells with liposome or ultrasound and microbubble. 24-48 hours later, the transfection efficiency and the concentrations of hBMP-2 were measured with fluoresence microscope and enzyme-linked immunosorbent assay (ELISA) respectively. The data were analyzed by curve fitting and t-test of SPSS 11.5. RESULTS: The transfection efficiency rate was (7.30 +/- 1.58)% in liposome group, compared with (11.77 +/- 3.16)% in ultrasound-mediated microbubble destruction group (P< 0.05). The concentration of hBMP-2 after transfection was (1164.35 +/- 724.67) pg/mL in liposome group, versus (2932.70 +/- 656.27) pg/mL in ultrasound-mediated microbubble destruction group (P<0.05). CONCLUSION: Ultrasound-mediated microbubble destruction could significantly improve the transfection efficiency and expression of hBMP-2 gene in NIH3T3 cells. It may provide a new and effective gene delivery system for gene therapy in periodontal regeneration.

[Gene therapy of bone morphogenetic protein-2 for periodontal tissue regeneration in vivo].

PURPOSE: To evaluate the effect of pIRES-rhBMP-2 gene on the regeneration of periodontal bone defects of beagle dogs in vivo. METHODS: Following surgery to create a U-shaped periodontal osseous defect on the buccal aspect of 6 mandibular premolar (P2,P3,P4) mesial roots, 6 adult beagle dogs were divided into three groups, pIRES-rhBMP-2 group, rhBMP-2 group and phosphate-buffered saline (PBS) group respectively. The animals were sacrificed after 4 or 8 weeks and the mandible taken for histological examination. Histometric measurements were performed with Image-Pro- Express system. The height of new bone and cementum and the formation of new connective tissue were analyzed and compared. The results were analyzed by N-K test using SAS6.12 software package. RESULTS: After 8 weeks, a complete osseous healing occurred and dense new periodontal ligament fibers rich in blood vessels were observed in the pIRES-rhBMP-2 group and the rhBMP-2 group, whereas fewer new bone occurred and sparse collagen fibers aligned irregularly were observed in the blank control group. The height of new bone and cementum and the formation of new connective tissue were significantly greater in the two experimental group than in the blank control group (P<0.05,P<0.01), whereas there was no significant difference in the two experimental groups (P>0.05). CONCLUSION: pIRES-rhBMP-2 can accelerate the regeneration of dogs' periodontal bone defects.

[Construction of a eukaryotic expression vector containing enhanced green fluorescence protein and recombinant human bone morphogenetic protein-2].

PURPOSE: To construct recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP containing enhanced green fluorescence protein (EGFP) and recombinant human bone morphogenetic protein-2 (rhBMP-2). METHODS: A pair of primers specific for amplifying the DNA fragment encoding rhBMP-2 were designed and synthesized. The targeted DNA fragment was amplified from pIRES-rhBMP2 by PCR.rhBMP-2 and EGFP were inserted to the proper sites of vector pIRES, and internal ribozyme entry site (IRES) sequence was between the genes coding for rhBMP-2 and EGFP. The recombinant plasmid pIRES-rhBMP2-EGFP was first propagated in E.coli DH5alpha, and then was confirmed to contain hBMP2 cDNA sequence by agarose gel electrophoresis and DNA sequence analysis. RESULTS: The construction of the recombinant eukaryotic dual-expression plasmid pIRES-rhBMP2-EGFP and the open reading frame were confirmed through restriction enzyme mapping analysis and DNA sequencing. CONCLUSION: By PCR, T/A cloning, the cDNA fragment encoding rhBMP2 and EGFP fragment can be cloned into pIRES to construct the recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP.

[Biological effects of tetracycline on cultured human periodontal fibroblasts].

OBJECTIVE: To explore the biological effects of tetracycline on cultured human periodontal ligament fibroblasts (HPDLFs). METHODS: Increasing concentrations of tetracycline (1, 5, 20, 100, 500, 2500 microg/ml) were added to the medium of cultured HPDLFs, respectively. After co-incubated for 2 days, cell morphology was observed under reverse microscope, meanwhile, cell proliferation activity was assayed using MTT, the total amount of protein was detected with Coumassie Bright Blue method and DNA synthesis was measured by 3H-TdR. RESULTS: Over a concentration range of 1 to 100 microg/ml, cells demonstrated a normal appearance, spindle or fusiform shaped. Moreover, at a concentration range of 20 to 100 microg/ml, tetracycline significantly enhanced the proliferating activity and biosynthesis of HPDLFs (P < 0.01). However, higher concentration (2500 microg/ml) not only changed cell morphology, but also significantly inhibited cellular activity. CONCLUSION: The results suggested that proper doses of tetracycline could promote proliferation and biosynthesis of HPDLFs while higher concentrations of tetracycline had cytotoxic effect.

[Effects of minocycline on biobehavior of human periodontal ligament cells in vitro].

OBJECTIVE: To evaluate the influence of minocycline on the proliferation and biosynthesis of human periodontal ligament cells (HPDLCs) in vitro. METHODS: Various concentrations of minocycline (1, 5, 20, 100, 500, 2 500 mg/L) were added to the medium of cultured HPDLCs respectively. After co-incubated for 2 days, cell morphology was observed under reverse microscope, cell proliferation activity was assayed using MTT, the total amount of protein was detected with Coumassie Bright Blue method and DNA synthesis was measured by (3)H-TdR. RESULTS: The presence of minocycline not exceeding 500 mg/L in the medium resulted in no morphological change of HPDLCs. Moreover, at a concentration range of 5 to 100 mg/L, minocycline significantly enhanced the proliferative activity and biosynthesis of HPDLCs (P < 0.01). However, higher concentration (2 500 mg/L) not only changed cell morphology under microscope, but also significantly inhibited cellular activity. CONCLUSIONS: The results suggest that proper doses of minocycline could promote biobehavior of HPDLCs, while higher concentrations of minocycline had cytotoxic effect which may intervene affect tissue repair and regeneration.

[The effect of bFGF on proliferation of periodontal fibroblast-like cells of dogs].

OBJECTIVE: To evaluate the effects of basic fibroblast growth factor(bFGF) on proliferation of periodontal fibroblast-like cells in vivo. METHODS: A U-shaped osseous defect was produced on the buccal side of the mesial root. Four posterior teeth were conducted in four quadrants. Each quadrant included 4 groups: control, bFGF, expanded polytetrafluoroethylene(ePTFE) membrane, bFGF + ePTFE. Each time the 4 teeth sites in one quadrant were operated weekly and each dog experienced 4 times of operations. Bromodeoxyuridine(BrdU) was injected 1 hour prior to sacrificing the dogs at 4 weeks after first surgery. Immunohistochemical method was applied to count the BrdU-labeled fibroblast-like cells. RESULTS: The number of BrdU-labeled cells reached the maximum at the 2nd week among all groups and then, decreased with time. Both bFGF and bFGF + ePTFE treated group had significantly more BrdU+ cells than remained control or ePTFE groups (P < 0.05) at 1st, 2nd weeks after surgery. CONCLUSION: 2 weeks after periodontal surgery is active phase of proliferation of periodontal fibroblasts. bFGF enhances fibroblast proliferation in early periodontal wound healing, and in turn accelerate periodontal regeneration.

[Biological effects of tetracycline and doxycycline on cultured human periodontal ligament cells].

OBJECTIVE: To explore the biological effects of tetracycline and doxycycline on cultured human periodontal ligament cells(HPDLCs). METHODS: Increasing concentrations of two drugs(1,5,20,100,500,2500 microg/ml)were added to the medium of cultured HPDLCs separately. After being co-incubated for 2 days, cell morphology was observed under reverse microscope; meanwhile, cell proliferation activity was assayed using MTT, the total amount of protein was detected by Coumassie Bright Blue method and DNA synthesis was measured by 3H-TdR. RESULTS: Over a concentration range of 1 to 100 microg/ml, cell morphology was normal. Moreover, at a concentration range of 20 to 100 microg/ml, tetracycline significantly enhanced the proliferative activity and biosynthesis of HPDLCs (P<0.01) while doxycycline significantly promoted DNA synthesis only at the concentration of 20 microg/ml. However, higher concentration of the two drugs (2500 microg/ml) not only changed cell morphology under microscope, but also significantly inhibited cellular activity. CONCLUSION: The results suggested that proper doses of tetracycline could promote proliferation and biosynthesis of HPDLCs,while doxycycline had limited effects on biobehavior of HPDLCs. Nevertheless, higher concentrations of the two drugs both had cytotoxic effect.