[Isolation, culture and ultrastructure analysis of mesenchymal stem cells from human periodontal ligament].

OBJECTIVE: To isolate and culture human periodontal ligament stem cells (PDLSCs) and observe its ultrastructure. METHODS: The proliferation and growth characteristics of human periodontal ligament cells were observed in primary culture and colony culture. PDLSCs were isolated by magnetic activated cell sorting (MACS) and ultrastructural characterization was observed by electron microscopy. RESULTS: When the cells were cultured at low density, PDLSCs grew in a colony-like manner. With the exception of a small amount of rough endoplasmic reticulum, ribosomes, and mitochondria, relatively few organelles were found in the cytoplasm, suggesting that they had remained undifferentiated. CONCLUSION: PDLSCs showed colony-like growth capacity and had ultrastructural characterization with stem cells. This indicated that PDLSCs could act as the appropriate seed cells for cell-based periodontal tissue regeneration.

[Effect of icariin on osteoblastic differentiation gene expression of human periodontal ligament cells].

OBJECTIVE: To observe the effect of icariin on human periodontal ligament cells (hPDLCs) differentiation to osteoblast gene expression. METHODS: The fifth generation of the cultured hPDLCs was added with the concentration of 0.01 mg/L icariin, and the added osteogenic medium used as blank control group, alizarin red staining of icariin on human periodontal ligament cells was observed for 21 days; the 2, 4, and 6 days of Q-PCR quantitative analysis of icariin on human periodontal ligament cells were made for osteogenesis gene alkaline phosphatase (ALP), type I collagen and osteocalcin (OC) gene expression. RESULTS: For the 21 days, alizarin red staining icariin group formed more mineralized nodules; on the 2nd, 4th, and 6th days, the group of icariin promoted the expression of ALP and OC mRNA, reached the peak value on day 6, compared with the control group with significant difference (20.15±6.67 vs. 7.90±0.71, 4.13±0.56 vs. 3.55±0.08, P<0.01). The second day, the highest expression of type I collagen appeared, then decreased gradually after, statistically compared with the control group (P<0.05). CONCLUSION: Icariin can promote the human periodontal ligament cells differentiation to osteoblast, and promote the osteogenesis gene expression.

[Effect of icariin on the expression of receptor activator nuclear factor-κB ligand and osteoprotegerin of human periodontal ligament cells].

OBJECTIVE: To investigate the effect of icariin on the expression of receptor activator nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) of human periodontal ligament cells (hPDLC) inhibited by sonicated extracts of Porphyromonas gingivalis (Pg). METHODS: hPDLC were extracted from 14 premolars extracted from 11 to 18-year-old patients for orthodontic purpose and cultured. The 7th passages of hPDLC were divided into six groups, each group was stimulated with 0, 0.001, 0.01, 0.1, 1 mg/L icariin and 50 mg/L sonicated extracts for 48 h. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of RANKL and OPG. RESULTS: Pg extracts group expressed more RANKL than the control group (3.60 ± 0.11 vs 1.08 ± 0.19), OPG expression decreased (0.32 ± 0.08 vs 1.01 ± 0.08), and RANKL/OPG ratio significantly increased (11.20 ± 0.13 vs 1.00 ± 0.10). Compared with control group, RANKL and OPG expression changed in groups stimulated with 0.001, 0.01, 0.1, 1 mg/L icariin. OPG expression was higher in 0.1 mg/L group than in the control (36.84 ± 0.05 vs 1.01 ± 0.08), RANKL/OPG ratio was significantly lower than the control group (0.04 ± 0.03 vs 1.00 ± 0.10) (P < 0.01). CONCLUSIONS: The concentration of 0.1 mg/L icariin can inhibit hPDLC expression of RANKL in sonicated extracts of Pg and promote OPG expression.

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

OBJECTIVE: To investigate the effects of emdogain, enamel matrix derivative (EMD), on the proliferation, cell cycle, mineralization and ultrastructure of human periodontal ligament (PDL) cells in vitro. METHODS: The influence of cell growth on PDL cells was evaluated by Cell Counting Kit-8 (CCK-8) in the presence and absence of emdogain, after 1, 3, and 5 d of culture. DNA synthesis and ultrastructure of PDL cells were observed by flow cytometry(FCM) and transmission electron microscopy (TEM) in the presence and absence of emdogain after 3 d of culture. The increasing of osteogenic capacity was verified by the expression changes of osteogenic differentiation markers of bone sialoprotein (BSP) and osteopontin (OPN) in emdogain-treated PDL cells by immunohistochemicl staining. RESULTS: Incubation of PDL cells with emdogain after 3 d significantly stimulated cell growth and DNA synthesis. Emdogain enhanced the osteogenic potential of PDL cells by high expression of osteogenic differentiation markers of BSP and OPN. CONCLUSION: The data indicate that Emdogain enhances cell proliferation and promotes differentiation of PDL cells, which contributes to periodontal tissue regeneration .

[Effect of sonicated extracts of Porphyromonas gingivalis on receptor activator of NF-κB ligand and osteoprotegerin expression in periodontal ligament cells].

OBJECTIVE: To evaluate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression in human periodontal ligament cells (HPDLC) and the effect of Pg on bone resorption in periodontitis. METHODS: HPDLC were exposed to 25, 50 mg/L sonicated extracts of Pg for 6 h, HPDLC without treatment served as control. The expression of RANKL-OPG mRNA and protein were examined by real time polymerase chain reaction and Western blotting. OPG protein in the supernatant was examined by enzyme linked immunosorbent assay (ELISA). The data were statistically analyzed by SPSS 13.0 and one-way analysis of variance (ANOVA). RESULTS: When HPDLC were exposed to sonicated extracts of Pg, the expression of RANKL mRNA and protein in 25 mg/L and 50 mg/L groups were higher than that of control group (P < 0.05), the expression of OPG mRNA in 50 mg/L group (0.087 ± 0.021) was lower than that of control group (0.240 ± 0.019) (P < 0.05), and OPG protein in 25 mg/L and 50 mg/L groups (0.813 ± 0.007, 0.398 ± 0.009) was lower than that of control group (1.131 ± 0.005) (P < 0.01). OPG protein expression in the supernatant was not significantly different between experimental group and control group. CONCLUSIONS: Sonicated extracts of Pg exposed to HPDLC can up-regulate RANKL expression, down-regulate OPG expression and influence bone metabolism.

Cell-cycle-dependent variations in the FTIR spectroscopy of HeLa cells treated with trichostatin A.

It is quite complex to evaluate the mechanism of action for antitumor drugs on cancer cells. Studies have pointed out that there is an unique advantage of Fourier transform infrared spectrum to obtain a fingerprint of all molecules present in the cells when cancer cells were exposed to anti-cancer drugs. Trichostatin A (TSA) is a most potent reversible inhibitor of mammalian histone deacetylases. It can inhibit cancer cell growth in vitro and in vivo. In the present study, HeLa cells were exposed to 0, 50, 100, 200, 300 and 400 nmol x L(-1) TSA, and FTIR spectra were applied to evaluate the effect of TSA on cancer cells. Results show that there is some significant relationship between the changes in FTIR absorption and cell cycle arresting. On the other hand, this investigation shows that the concentration of TSA had to be more than 200 nmol x L(-1) in order to ensure A1080 cm(-1)/A1540 cm(-1) > or = 1 for inhibiting cell proliferation.

[FTIR spectroscopic analysis of HeLa cell irradiated by low-energy ions].

In the present paper, low-energy N+ ions produced by low-energy ionic radiometer were used to simulate low-energy ions in astrospace implanting HeLa cell. Then, the effect and mechanism of low-energy ion on the cell were studied with FTIR spectroscopic analysis. In this study, low-energy ions were produced and accelerated in vacuum, and cells would be affected by vacuum when they were implanted by low-energy ions, so mineral oil was used to protect cells from water evaporation. Cells were collected after being implanted, then the change in the content and constructional form in cellular macromolecule with infrared spectrometry. Result indicated that the spectroscopic peak position of differently worked cells was obviously different as compared to the control cell (3300 cm(-1)). Spectroscopic peak position of all samples except implanting 5 x 10(14) N+ x cm(-2) removed to longer wavelength, and the peak position in vacuum of 2 x 10(15) N+ x cm(-2) sample moved to 3420 cm(-1). In addition, the 1378 and 2360 cm(-1) spectroscopic peak positions in control cell all moved to longer wavelength in every worked group. In a word, FTIR spectroscopic analysis indicates that low-energy ion implantation could arouse change in nucleic acid or protein in HeLa cell.

[Absorption spectrum study of HeLa cells treated with vacuum and low-energy ions implantation].

Mineral oil was selected to protect HeLa cells from water evaporation during low-energy ions implantation in the present paper. Then, HeLa cells having been treated with vacuum and low-energy N+ ions implantation were used to collect ultraviolet absorption spectrum by spectrophotometer. Analytical results indicated that HeLa cells had some characteristic absorption peaks near 202 and 260 nm, respectively. And then the study also found: (1) The spectral intensity increased with the vacuum treatment time. In addition, the effect of vacuum on cellular spectrum was greater than that of mineral oil. (2) The influence of low energy N+ ions on absorption spectrum was far more than that of vacuum. (3) The spectral intensity increased with the implantation dose. According to these results, the effect of low-energy N+ ions implantation and vacuum on tumorous cells (HeLa cells), especially on the molecular configuration and component of tumorous cells (HeLa cells) was discussed. In a word, this study provides a basis for further research on the functionary mechanism of low-energy ions implantation on biomaterial.

[Study on absorption spectra of cell substrate for Hep-2 cell after being radiated by X-ray].

UV-absorption spectra of the Hep-2 cell's culture medium RPMI1640 (10% Foetal Calf Serum) were collected by UV-3101 spectrophotometer after the Hep-2 cell was radiated by X-ray and cultivated for 24, 48 and 72 h, and the absorbability of the proteins in the substrate was analyzed. From these results it was found that there were visible differences among these absorption spectra. In particular, the absorption peaks of the RPMI1640 culture medium during the cultivation shifted from 233 to 235 nm, while the absorption peak at 278 nm became more and more smooth and even finally disappeared with the cultivation time. On the other hand, the absorption intensity of the different-dose groups rose greatly with the time, and were all lower than the control group until the cells were cultivated for 72 h after being radiated by X-ray. It was showed that the content of each amino acid has already changed. That is, during the growing course of the cancer cells, the tryptophan and casein were not depleted equivalently. And there were some important relations between the absorption spectra and the cells' apoptosis and necrosis induced by X-ray. This will be a foundation for the study of the best X-ray dose for the larynx carcinoma.

[Preliminary study on anti-periodontitis immunization with DNA vaccine].

OBJECTIVE: To observe the protection against periodontal bone loss in the Sprague-Dawley (SD) rats periodontitis model, with the recombined plasmid pcDNA3.1+/kgpcd as DNA gene vaccine. METHODS: PcDNA3.1+/kgpcd was delivered into rats by submandibular gland-targeted injection. The anti-KGPcd sIgA in saliva was measured by indirect ELISA method. Immunohistochemistry staining was used to assess the protection in the animal model. RESULTS: The level of specific anti-KGPcd sIgA in saliva of the experimental group was significantly higher than that of control group. HE staining showed that immunization with recombined plasmid pcDNA3.1+/kgpcd could protect or minimize tissue destruction caused by subsequent P. gingivalis challenge in the rat model. CONCLUSIONS: The results indicate that pcDNA3.1+/kgpcd was a good candidate for anti-periodontitis gene vaccine and could provide protection against Porphyromonas gingivalis-caused periodontitis in rat lesion model.

[Effect of cancer cells on the spectra of serums].

This article studied the different effects on the spectra of serums by adding different amount of cancer cells into serum, and gives the comparison between CNE cells' serum spectra and Hela' as well as that between abnormal serums and normal ones. Experiment results showed that the intensity of the absorption spectra and the fluorescence spectra exhibits a trend that it increases with the decrease in the cancer cells' number, while begins to drop when it goes up to a certain extent. The intensity of the normal serums' spectra is obviously higher than that of the abnormal one. It is concluded that the changes in the intensity of fluorescence spectra' show that the cancer cells restrain the vibration of some group in the normal serums, and the absorption spectra' reveal that the cancer cells prevent the casein from absorbing light.

[The study of absorption spectrum for cell substrate].

The authors collected the absorption spectrum of RPMI 1640 and DMEM substrates that cultivated Hela and CNE by UV-3101 spectrophotometer and analysed the absorbability of proteins in the substrate. The absorption peaks of the RPMI 1 640 culture medium that cultivated cells for different times shifted from 227 to 222 or 218 nm and from 278 to 280 nm respectively; while during growing course of cultivated cells, one of the absorption peaks of DMEM culture medium shifted from 224 nm to one near 221 nm, and the absorption peak 278 nm almost had no shift. All of these shifts show that the content of each amino acid such as tryptophan and casein has already changed. That is, during the growing course of cultivating cancer cells, the tryptophan and casein were not depleted equivalently. In the growth period of Hela and CNE, they consumed different amino acid. So they need different component proportion for amino acid.

[Construction of eukaryotic expression vector for KGPcd gene from Porphyromonas gingivalis and expression in mammalian cells].

OBJECTIVE: This study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells. METHODS: Eukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method. RESULTS: It proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA. CONCLUSION: The eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.