A polyglutamic acid/tannic acid-based nano drug delivery system: Antibacterial, immunoregulation and sustained therapeutic strategies for oral ulcers.

Oral ulcers are a common inflammatory mucosal ulcer, and the moist and dynamic environment in the oral cavity makes topical pharmacological treatment of oral ulcers challenging. Herein, oral ulcer tissue adhesion nanoparticles were prepared by using esterification reaction between polyglutamic acid and tannic acid, and at the same time doxycycline hydrochloride was loaded into the nanoparticles. The obtained slow drug release effect of the drug-loaded nanoparticles reduced the toxicity of the drug, and by penetrating into the fine crevice region of the wound tissue and adhering to it, they could in-situ release the carried drug more effectively and thus have shown significant antibacterial effects. In addition, tannic acid in the system conferred adhesion, antioxidant and immune regulation activities to the nanocarriers. A rat oral ulcer model based on fluorescent labeling was established to investigate the retention of nanoparticles at the ulcer, and the results showed that the retention rate of drug-loaded nanoparticles at the ulcer was 17 times higher than that of pure drug. Due to the antibacterial and immune regulation effects of the drug-loaded nanoparticles, the healing of oral ulcer wounds was greatly accelerated. Such application of doxycycline hydrochloride loaded polyglutamic acid/tannic acid nanoparticles is a novel and effective treatment strategy for oral ulcer.

A Nature-Inspired Versatile Bio-Adhesive.

The application of most hydrogel bio-adhesives is greatly limited due to their high swelling, low underwater adhesion, and single function. Herein, a spatial multi-level physical-chemical and bio-inspired in-situ bonding strategy is proposed, to develop a multifunctional hydrogel bio-glue using polyglutamic acid (PGA), tyramine hydrochloride (TYR), and tannic acid (TA) as precursors and 4-(4,6-dimethoxytriazine-2-yl) -4-methylmorpholine hydrochloride(DMTMM) as condensation agent, which is used for tissue adhesion, hemostasis and repair. By introducing TYR and TA into the PGA chain, it is demonstrated that not only can the strong adhesion of bio-glue to the surface of various fresh tissues and wet materials be realized through the synergistic effect of spatial multi-level physical and chemical bonding, but also this glue can be endowed with the functions of anti-oxidation and hemostasis. The excellent performance of such bio-glue in the repair of the wound, liver, and cartilage is achieved, showing a great potential in clinical application for such bio-glue. This study will open up a brand-new avenue for the development of multifunctional hydrogel biological adhesive.

A bionic composite hydrogel with dual regulatory functions for the osteochondral repair.

Due to the avascular nature of cartilage, it is difficult to heal and regenerate spontaneously after injury. At present, tissue engineering has become a promising strategy for repairing damaged cartilage, but the use of seed cells and growth factors is limited. In addition, the importance of mechanical compatibility of scaffold materials is often ignored. In this study, osteochondral scaffold was designed as a bilayer structure with a dense γ-Polyglutamic acid/carboxymethyl chitosan/bacterial cellulose (PGA/CMCS/BC) hydrogel cartilage layer and a porous nano HA-containing PGA/CMCS/BC hydrogel osteogenic layer. In addition, bioactive ions were introduced into the hydrogel scaffold to adjust the mechanical and swelling properties of the material to match the mechanical properties of natural articular cartilage. At the same time, based on the structural characteristics of bone and cartilage, magnesium and copper ions were introduced into the double-layer hydrogel scaffold, respectively, to prepare the cartilage layer and the bone layer, which endowed the material with excellent antibacterial properties and achieved the purpose of the integrated repair of bone and cartilage. The results showed that, after adding magnesium ions, the tensile breaking strength of material was increased from 0.66 MPa to 1.37 MPa，the corresponding compression modulus of the material (strain 0-12%) increased from 0.15 MPa to 0.58 MPa whilst the maximum mass swelling rate decreased from 155% to 75%. The results of in vivo experiments show that the group with bioactive ions had a much better effect on the repair of osteochondral defects, compared with group without bioactive ions, demonstrating such double ion regulation strategy is a very practical strategy for the treatment of osteochondral defects.

Piezoelectric Effect of Antibacterial Biomimetic Hydrogel Promotes Osteochondral Defect Repair.

The lack of vascular tissue and the low metabolism and biological activity of mature chondrocytes lead to the low regeneration ability of articular cartilage. People try to solve this problem through various methods, but the effect is not very ideal. Inspired by the piezoelectric effect of collagen in cartilage tissue, this work focused on the design of a biomimetic hydrogel by introducing piezoelectric materials and silver nanowires into hydrogel to endow them with piezoelectric and antibacterial properties to promote tissue regeneration. Additionally, the mechanical and swelling properties of the material were adjusted to match natural articular cartilage. Based on bionic principles, a double-layer piezoelectric hydrogel was prepared and applied for the repair of osteochondral defects. An enhanced repair effect of osteochondral defects has been seen, which has demonstrated potential values for future application in bionics principle- and piezoelectric effect-based osteochondral tissue engineering. Furthermore, piezoelectric effect-induced degradation was observed. These results fully indicated the positive effect of the piezoelectric effect on promoting the regeneration of osteochondral tissue and in vivo degradation of materials.

By Endowing Polyglutamic Acid/Polylysine Composite Hydrogel with Super Intrinsic Characteristics to Enhance its Wound Repair Potential.

In this study, multifunctional porous composite hydrogels are prepared via composite technology and using poly(glutamic acid) and (polylysine) as precursors, casein as foaming agent, and calcium ions as coagulant. The results show that the as-prepared hydrogels have high porosity and water absorbency, and good mechanical, inherent antibacterial, and bioadhesive properties. The results show such high water absorption, bioadhesion, and porosity of the as-prepared hydrogel can effectively concentrate blood components and seal wounds better. The release of calcium ions in the as-prepared hydrogels can activate coagulation factors. Both factors can play an important role in hemostasis. The excellent hydroscopicity, moisture retention, adhesion, and inherent antibacterial properties of the as-prepared hydrogel can create a moist, sterile, and closed microenvironment for the wound healing. The experimental results of a deep skin defect model have verified its good effect of promoting wound repair. These inherent excellent properties can endow the as-prepared hydrogel with a wide range of application values.

Constructing Gene-Enhanced Tissue Engineering for Regeneration and Repair of Osteochondral Defects.

In situ sustained release of endogenous growth factors from cells is a challenge for repair and regeneration of tissue. Although recombinant adenovirus vectors are an effective delivery system that can prolong the release of growth factors and is very suitable for the therapy of growth factors, these recombinant adenovirus vectors that are widely used at present have low safety and stability in terms of long-term expression. In this study, the above problems are solved by knocking out both E1 and E3 genes at the same time and directly inserting the gene fragments encoding target proteins after the inverted terminal repeats. Finally, the combination of gene therapy with tissue engineering in regeneration and repair of full-thickness defects of osteochondral tissue are applied as an example. The results show that this strategy can achieve complete repair of articular osteochondral defects and recovery of their function, and meanwhile solve the problems of low safety and expression instability of recombinant adenovirus vectors. This method provides a bright prospect for the application of gene enhanced tissue engineering in the regeneration and repair of joint tissue, and also provides a reference for the repair and regeneration of other tissues.

Study of bilineage differentiation of human-bone-marrow-derived mesenchymal stem cells in oxidized sodium alginate/N-succinyl chitosan hydrogels and synergistic effects of RGD modification and low-intensity pulsed ultrasound.

The level of formation of new bone and vascularization in bone tissue engineering scaffold implants is considered as a critical factor for clinical application. In this study, an approach using an RGD-grafted oxidized sodium alginate/N-succinyl chitosan (RGD-OSA/NSC) hydrogel as a scaffold and low-intensity pulsed ultrasound (LIPUS) as mechanical stimulation was proposed to achieve a high level of formation of new bone and vascularization. An in vitro study of endothelial and osteogenic differentiations of human-bone-marrow-derived mesenchymal stem cells (hMSCs) was conducted to evaluate it. The results showed that RGD-OSA/NSC composite hydrogels presented good biological properties in attachment, proliferation and differentiation of cells. The MTT cell viability assay showed that the total number of cells increased more significantly in the LIPUS-stimulated groups with RGD than that in the control ones; similar results were obtained for alkaline phosphatase activity/staining and mineralized nodule formation assay of osteogenic induction and immunohistochemical test of endothelial induction. The positive synergistic effect of LIPUS and RGD on the enhancement of proliferation and differentiation of hMSCs was observed. These findings suggest that the hybrid use of RGD modification and LIPUS might provide one approach to achieve a high level of formation of new bone and vascularization in bone tissue engineering scaffold implants.

[Primary culture and identification of rat glomerular podocytes].

OBJECTIVE: To establish an easy and feasible method for primary culture and identification of rat glomerular podocytes. METHODS: Glomeruli from Sprague-Dawley (SD) rats weighing 60-100 gram were isolated by the method of different size combination of screen. Isolated glomeruli were appropriately digested with 2 g/L type IV collagenase and cultured in 25 cm2 plastic flask coated with rat tail collagen in K1-3T3 medium with ITS-X (containing insulin-transferrin-selenium). Subculture of primary cultured epithelial cells was performed at 9-10 days after implantation of collagenase digested glomeruli. Podocytes were identified by the morphology study with scanning electron microscope and inverted microscope, as well as the immunohistochemistry staining (SP methods) study for the expression of keratin, desmin and Wilms' tumor suppressor-1 (WT-1). RESULTS: Epithelial cells outgrowth from isolated glomeruli appeared after 3 days primary culture and grew to confluence with cobblestone-appearance at 9-10 days. These cobblestone cells were subcultured at this point and gradually conversed into large, flat arborized cells with well-developed processes and microvilli. These arborized cells were negative expression with desmin staining and showed positive expression of cytokeratin and WT-1, which indicated that they were podocytes. CONCLUSION: Implantating collagenase digested-glomeruli is an easy and feasible method for primary culture of rat glomerular podocytes. WT-1 may serve as a good marker to identify rat glomerular podocytes.

Synthesis of an RGD-grafted oxidized sodium alginate-N-succinyl chitosan hydrogel and an in vitro study of endothelial and osteogenic differentiation.

Hydrophilic surfaces for hydrogels as bone tissue engineering scaffolds are not beneficial for the adsorption of protein and not conducive to the adhesion and growth of cells. In this study, we proposed to use an oxidized sodium alginate-N-succinyl chitosan hydrogel as a bone tissue engineering scaffold material and to overcome this issue by using RGD to modify this kind of hydrogel. The physicochemical properties of the obtained hydrogels were characterized and an in vitro endothelial differentiation and osteogenic differentiation study of bone-marrow-derived mesenchymal stem cells (BMSCs) was conducted to evaluate it. The results showed that the RGD-grafted oxidized sodium alginate-N-succinyl chitosan hydrogel not only had a good degradability but also enhanced cell adhesion and proliferation and promoted endothelial differentiation and osteogenic differentiation of BMSCs. Based on the results, it can be expected that RGD-grafted oxidized sodium alginate-N-succinyl chitosan hydrogel might be an optimal material for bone tissue engineering scaffold whenever it is used alone, or composed with other materials in the future.

BnRCH gene inhibits cell growth of Hela cells through increasing the G2 phase of cell cycle.

The ubiquitin-proteasome pathway (UPP) is an important protein degradation system universally existing in eukaryotic organisms from yeast to human. In this system, hundreds of E3 ubiquitin-protein ligases are most important because they provide the substrate specificity and control many cellular processes. UPP has been found to be relevant to cancer development. BnRCH, the protein product from a novel gene isolated from Brassica napus, also has E3 ubiquitin-protein ligase activity. In order to exploit its potential use, human cervical carcinoma cell Hela (Hela cells) was transiently and stably transfected with BnRCH. The experimental results demonstrated: (1) in Hela cells, BnRCH inhibited the cell growth of Hela cells and increased their sensitivity to the anti-cancer chemotherapeutic drug cisplatin; and (2) the growth inhibition effect of BnRCH in Hela cells was found due to G2 phase cell cycle arrest with the transcriptional up-regulation of p21 (waf1/cip1), rather than apoptosis. This research suggests BnRCH has potential use in cancer therapy.

[Influence of mutation of -1997G-->T of COL I A1 gene on the biochemical function of osteoblast].

OBJECTIVE: To study the influence of mutation of -1997G-->T of COL I A1 gene on the biochemical function of osteoblast, and the pathomechanism of BMD. METHODS: Spongy bones were obtained to culture osteoblast primarily during total hip or knee replacements. The genotype of osteoblast was identified with PCR-RFLP. The amount of mRNA of COL I A1 and collagen type I were determined by RT-PCR and ELISA. The growth of osteoblast, the activity of bone ALP, and the amount of calcium in cell matrix and calcium nodus were measured. RESULTS: Three genotypes GG, GT and TT in osteoblast were successfully identified. Compared with GG and GT genotypes, lower expression of mRNA of COL I A1 gene, lesser collagen type I, calcium in cell matrix, and calcium nodus were found in the cells with TT genotype (P < 0.01). No significant differences were found between GG and GT genotype (P > 0.05). There were no significant differences in age, growth of osteoblast, and activity of bone ALP among the three genotypes (P > 0.05). CONCLUSION: Cells with TT genotype have low expression of mRNA of COL I A1 gene and less collagen type I , calcium in cell matrix and calcium nodus. The lower amount of collagen type I synthesized by osteoblast can decrease the matrix outside the bone cells and result in insufficient site for calcium deposition. This may be the cause of lower BMD in patients with TT genotype.

[Treatment of early avascular necrosis of femoral head by core decompression and transplantation of human hepatic growth factor gene-modified osteoblasts: experiment with rabbits].

OBJECTIVE: To evaluate the effects of transplantation of human hepatocyte growth factor (hHGF) gene-modified osteoblasts combined with core decompression in treatment of avascular necrosis of femoral head (ANFH). METHODS: The plasmid pcDNA3.1(+)-hHGF containing hHGF gene was constructed. Osteoblasts were isolated from fetal rabbits, cultured, and transfect3d with the plasmid pcDNA3.1(+)-hHGF or blank plasmid pcDNA3.1(+), or used as controls. Thirty-six adult New Zealand rabbits were made into ANFH models, underwent core decompression, and were randomly divided into 3 groups. Group A, transplanted with osteoblasts transfected with pcDNA3.1(+)-hHGF plasmid, Group B, transplanted with osteoblasts not transfected with pcDNA3.1(+)-hHGF plasmid, and Group C, injected with PBS medium. 2, 4, and 8 weeks later samples of femoral head were obtained to undergo CT, histological examination, and capillary ink infusion so as to observe the angiogenesis and osteogenesis. RESULTS: The pcDNA3.1(+)-hHGF transfected osteoblasts showed stable expression of hHGF. The numbers of newly formed vessels of the femoral heads of the group transfected with pcDNA3.1(+)-hHGF-transfected osteoblasts 2 and 4 weeks later were (29.47 +/- 1.64) and (34.02 +/- 1.72)/cm2 respectively, both significantly higher than those of the group transfected with blank plasmid-transfected osteoblasts [(20.61 +/- 1.91) and (25.57 + 2.20)/cm2 respectively, both P <0. 01]. Eight weeks later the numbers of mature trabecular bone and bone marrow of Groups A and B were significantly higher than those of Group C. CONCLUSION: Core decompression combined with transplantation of HGF gene-modified osteoblasts promotes angiogenesis, enhances bone formation, and improves the restoration of avascular necrosis of femoral head.

[Construction and expression of recombinant plasmid pcDNA3.1(+) -hHGF available in osteoblast].

OBJECTIVE: To construct a plasmid carrying human hepatocyte growth factor (hHGF) gene and determine the effects of the hepatocyte growth factor (HGF) gene on proliferation and differentiation of osteoblast in vitro. METHODS: The full length cDNA of hHGF, which was amplified from human liver mRNA by RT-PCR, was cloned into pcDNA3.1(+) vector to construct pcDNA3.1(+) -hHGF recombinant plasmid. With lipofectamine, the recombinant plasmid pcDNA3.1(+) -hHGF was used to transfect the culture osteoblasts. After pcDNA3.1(+) - hHGF transfection, the positive cell clones were selected with G418. The stable transfection and expression of hHGF in the osteoblasts were measured by immunohistochemical staining and RT-PCR respectively. The effect of pcDNA3.1(+) -hHGF transfection on osteoblast proliferation was measured by MTT colorimetric assay. Flow cytometer was used to determine the effect of pcDNA3.1(+) -hHGF transfection on cell cycle of osteoblast. The quantitive detection of hHGF expression was performed through ELISA. Alkaline phosphatase (AP) was also detected using enzyme kinetics. RESULTS: The recombinant plasmid pcDNA3. 1(+) -hHGF was identified by restriction endonuclease digestion and nucleotide sequencing. Osteoblasts could be effectively transfected by pcDNA3.1(+) -hHGF with lipofectamine in vitro. The stable expression of hHGF in pcDNA3.1(+) -hHGF transfection osteoblast was confirmed. Human HGF protein was detected by immunohistochemical staining and RT-PCR in osteoblasts 4 weeks after cell clone selected with G418. pcDNA3.1(+) -hHGF gene transfer responsible to improve the proliferation was proved by MTT assay, flow cytometer showed cellular proportion in "S" phase obviously increased. AP activity in transfected cells was increased significantly. CONCLUSIONS: Osteoblasts which express stable and high-level hHGF was established successfully, exogenous hHGF gene could stimulate the proliferation, differentiation and function of osteoblast and could be applied further to gene therapy.

[Inhibitory effects of deoxyribozyme on the expression of Period1 gene in vitro and on morphine-induced psychic dependence in mice].

OBJECTIVE: To investigate the regulatory effects of deoxyribozyme on the expression of Period 1 gene in vitro and on the morphine-induced psychic dependence in mice. METHODS: The specific deoxyribozymes toward Period 1 mRNA was designed by MFold analysis and synethsized chemically. By LipofectAMINE mediated DNA transfection technique, DRz164 and pcDNA3-Per1 were introduced into NIH3T3 cells. The effects of deoxyribozyme on Period 1 gene were studied by reverse transcript-polymerase chain reaction (RT-PCR) and flow cytometry(FCM). The morphine-induced reward in mice was observed in a conditioned place preference test after pretreatment of the mice with the intracerebroventricular administration of deoxyribozyme. RESULTS: After NIH3T3 cells were transfected by pcDNA3-Per1 and DRz164, the Period 1 mRNA was reduced by 42.4%. And PERIOD proteins were decreased by 57.5%. After being pretreated with deoxyribozyme, the mice did not show morphine-induced place preference. CONCLUSION: DRz164 can highly block the expression of Period 1 gene, which cleaves the Period 1 mRNA in the transfected cells specifically. The suppression of morphine-induced place preference can be effected by pretreating the mice with alleviating their psychic dependence on morphine.

[Inhibitory effect of human brain myelin basic protein on H2O2-induced apoptosis of human lung cancer cell line YTLMC-90].

BACKGROUND & OBJECTIVE: Human brain myelin basic protein (MBP) distributes in nervous system and other tissues extensively, and can be detected in many kinds of tumor cells, such as lung cancer, breast cancer, and neuroglioma. However, it has not been reported whether MBP is relevant to the activity of neural invasion of tumors and whether MBP plays a role in biological behaviors of human lung cancer cells. This study was to investigate the inhibitory effect of MBP on hydrogen peroxide (H2O2)-induced apoptosis of human lung cancer cell line YTLMC-90. METHODS: YTLMC-90 cells were transfected with plasmid pSVCEPMBPCAT containing MBP cDNA minigene (test group), or empty vector pSVCEPCAT, or received no transfection (control group), and exposed to H2O2. The expression of MBP in YTLMC-90 cells was detected by Western blot. Cell proliferation was measured by MTT assay. The morphologic and ultra-structural changes of apoptotic cells were observed by microscopy with fluorescent staining of acridine orange (AO) and electron microscopy. The DNA fragmentation was examined by agarose gel electrophoresis. RESULTS: After exposed to 200 micromol/L H2O2 for 24 h, the inhibitory rate of cell growth was significantly lower in test group than in empty vector group and control group (36.67% vs. 78.67% and 84.00%, P<0.001). The morphologic and biochemical changes of apoptotic cells, such as shrinkage of cytoplasm and nucleus, fragmentation of chromatin, and ladder pattern of DNA, were commonly observed in cells in control group, but these apoptotic features were not discovered in test group. CONCLUSION: MBP markedly inhibits H2O2 cytotoxicity to YTLMC-90 cells through promoting cell proliferation and antagonizing H2O2-induced apoptosis.

Deoxyribozymes inhibit the expression of period1 gene in vitro.

To investigate the effect of two deoxyribozymes targeting period1 (per1) mRNA in vitro for exploring a novel gene therapy approach about circadian rhythm diseases, the specific deoxyribozymes targeting per1 were designed and synthesized chemically following MFold analysis according to its mRNA secondary structure. per1 RNA fragments were prepared by in vitro transcription of pcDNA3.1(+)-per1(164:256). The cleavage reactions containing deoxyribozymes and per1 RNA fragments were performed under certain conditions. With the transfection technique mediated by LipofectAMINE, pcDNA3-per1 and DRz164 or DRz256 were introduced into NIH3T3 cells. The effects of deoxyribozymes on per1 were studied by reverse transcript-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). When deoxyribozymes and RNA transcripts were incubated under the adopted conditions at 37 degrees C for 2 h, about 63% of per1(164:256) RNA transcripts were cleaved by DRz164 and about 50.5% by DRz256. After cotransfecting pcDNA3-per1 with DRz164 or DRz256, the expression of per1 mRNA was decreased, as indicated by RT-PCR semi-quantity analysis. FCM analysis showed that Per1 protein was inhibited. Both DRz164 and DRz256 targeting per1 have the specific cleavage activity toward per1 mRNA in vitro and can highly block the expression of per1 gene in cellular milieu.

[Feasibility of cationic lipid mediated nm23-h1 plasmid transfecting adnoid cystic carcinoma in vivo].

OBJECTIVE: To investigate the feasibility of plasmid nm23-h1 transfection on high metastatic potential adnoid cystic carcinoma (ACC-M) cell line mediated by cationic lipid. METHODS: ACC-M cell were implanted in the maxillofacial region in each of 40 BALB/c nude mice. After the tumor growth to 1 cm in diameter, 0.1 ml Lipofctamine-nm23-hl plasmid complex were injected intratumorally in 10 mice, 3 days after the first injection, 10 mices injected for twice, 10 mice as plamid-blank control, another 10 mice were injected 0.2 ml complex, 2, 3, 7days after the injection, the mice were killed and the specimen for HE and immunohistological chemistry study. RESULTS: nm23-h1 expression initiated in the tumor cells 3 days after the complex injection, 7 days later, the expression level increased accompanying with extracellular matrix increase, twice injection and multiple channel injection would gain better nm23-h1 expression than once injection and single-channel injection respectively. CONCLUSION: Cationic lipid mediated nm23-h1 plamid transfecting adnoid cystic carcinoma can gain small range positive expression, but the results give little prospect for further clinical treatment in such a manner.

[Impact of p16INK4A and p15INK4B on human hepatocellular carcinoma cell proliferation and apoptosis].

OBJECTIVE: Both tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene. METHODS: After identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry. RESULTS: Neither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene. CONCLUSION: p15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.

[The inhibitory effect of total flavonoids of hippophae on the activation of NF-kappa B by stretching cultured cardiac myocytes].

OBJECTIVE: To investigate the effect of total flavonoids of hippophae (TFH) on the improvement in myocardial hypertrophy. METHODS: Immunohistochemical method was used to assess the inhibitory effect of TFH on the activation of NF-kappa B by stretching cultured cardiac myocytes. RESULTS: It was found that NF-kappa B was activated by stretching cardiac myocytes in 10 hours; that TFH at the concentration of 1:400 partly inhibited the activation of NF-kappa B by stretching in cardiac myocytes; that TFH at the concentration of 1:200 and 1:100 completely inhibited the activation of NF-kappa B by stretching in cardiac myocytes. CONCLUSION: The results supported that the blockade of activation of NF-kappa B might be a potential access to the improvement in myocardial function with the use of TFH for treatment of hypertension and chronic cardiac insufficiency.

[Suppressive effect of brain derived neurotrophic factor on H2O2-induced apoptosis in human lung cancer cell YTMLC-90].

BACKGROUND & OBJECTIVE: It has been shown that neurotrophic factors such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin (NT-3/4) are synthesized in a variety of cells inside and outside the nervous system. These factors are not only able to promote neural survival, proliferation and apoptosis of neural cells but also relevant to the activity of neural invasion of tumors. It has not been reported to date whether BDNF may play roles in the biological behavior of human lung cancer cells. The aim of this experiment was to investigate the effect of BDNF on hydrogen peroxide (H2O2)-induced apoptosis in the human lung cancer cell line YTMLC-90. METHODS: The minigene pSVCEPBFCAT containing the promoter and enhancer elements of the human a1(I) collagen gene(COLIA1) at its 3' terminus followed by hBDNF gene cDNA was transfected and derived BDNF ectopic expression in the human lung cancer cells. The cell proliferation was measured by MTT assay. The morphological and ultra-structural changes of apoptotic cells were observed by microscopy with fluorescent stain of acridine orange and electron microscopy. The DNA fragmentation was examined by agarose gel electrophoresis. RESULTS: After exposure of growing cells to 200 micromol/L H2O2 for 24 hours, the inhibition rate of cell growth was 30% in the pSVCEPBFCAT-transfected YTMLC-90, 84.60% in controls of non-transfected YTMLC-90, and 80.00% in pSVCEPCAT-transfected YTMLC-90, respectively (P< 0.001). The morphological and biochemical changes of apoptotic cells such as shrinkage of cytoplasm and nucleus,fragmentation of the chromatin, and ladder pattern of DNA were commonly observed in the cell population of controls, but these apoptotic features were not discovered in the pSVCEPBFCAT-transfected YTMLC-90. CONCLUSION: BDNF markedly inhibits H2O2 cytotoxicity on human lung cancer cell YTMLC-90 by promoting YTMLC-90 proliferation and antagonizing H2O2-induced apoptosis.