[The Study of Recombinant Interfering Lentiviruses and Overexpressed Adenovirus Vectors Targeting Human c-Cbl Gene： Construction, Identification and Efficacy].

OBJECTIVE: To construct recombinant lentivirus and adenovirus which regulate the expression of c-Cbl gene and evaluate their efficacy. METHODS: The interference lentivirus and overexpressed adenovirus targeting human c-Cbl gene were constructed by gene recombination technology. Quantitative PCR and western blotting were used to detect the expression changes in c-Cbl gene and its transcription after leukemia cells (HL60,THP1) were infected by virus. RESULTS: Three recombinant interfering lentiviral vectors targeting human c-Cbl genes to successfully constructed and were identified by DNA sequencing, and the titers of the packaged viruses were all greater than 1×108 TU/ml. Among them, shRNA-2 lentivirus had the highest interference efficiency, and the expression of c-Cbl gene and CBL protein were decreased about 95% and 60% respectively after leukemia cells were infected with shRNA-2; In addition, the recombinant overexpression adenovirus targeting human c-Cbl gene was packaged successfully with the virus titer greater than 1×109 TU/ml. When leukemia cells were infected with adenovirus, the expression of c-Cbl gene and CBL protein were up-regulated about 10 times and 1.5 times respectively. CONCLUSION: Both recombinant interfering lentivirus and overexpression adenovirus can efficiently infect leukemia cells and affect the expressions of c-Cbl gene and CBL protein. It will lay a preliminary foundation for the subsequent study on the function of c-Cbl gene in tumor cells.

Sequencing and analysis of the complete mitochondrial genome of Ochotona hyperborea from China and its phylogenetic analysis.

The complete mitogenome sequence of Ochotona hyperborea was determined using long PCR. The genome was 17,063 bp in length and contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, one origin of L strand replication, and one control region. The overall base composition of the heavy strand is A (31.1%), C (28.7%), T (26.3%), and G (13.9%). The base compositions present clearly the A-T skew, which is most obvious in the control region and protein-coding genes. Mitochondrial genome analyses based on MP, ML, NJ, and Bayesian analyses yielded identical phylogenetic trees. This study verifies the evolutionary status of Ochotona hyperborea in Ochotonidae at the molecular level. The mitochondrial genome would be a significant supplement for the Ochotona hyperborea genetic background. The eight Ochotona species formed a monophyletic group with the high bootstrap value (100%) in all examinations.

Sequencing and analysis of the complete mitochondrial genome of Micromys erythrotis from China and its phylogenetic analysis.

The complete mitogenome sequence of Micromys erythrotis was determined using long PCR. The genome was 16,238 bp in length and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, 1 origin of L strand replication and 1 control region. The overall base composition of the heavy strand is A (33.7%), C (24.8%), T (29.1%) and G (12.4%). The base compositions present clearly the A-T skew, which is most obviously in the control region and protein-coding genes. Mitochondrial genome analyses based on MP, ML, NJ and Bayesian analyses yielded identical phylogenetic trees. This study verifies the evolutionary status of Micromys erythrotis in Muridae at the molecular level. The mitochondrial genome would be a significant supplement for the Micromys erythrotis genetic background.

Investigation and comparison of the protective activities of three functional proteins-lactoferrin, α-lactalbumin, and ß-lactoglobulin-in cerebral ischemia reperfusion injury.

The objective of this study was to evaluate the protection conferred by lactoferrin, α-lactalbumin, and ß-lactoglobulin in cerebral ischemia reperfusion (I/R) injury. Rat pheochromocytoma (PC12) cells were used to construct an oxygen and glucose deprivation model in vitro, and ICR mice underwent carotid artery "ligation-relaxation" to construct a cerebral I/R injury model in vivo. The levels of toll-like receptor 4 (TLR4) and downstream factors including nuclear factor-κB, tumor necrosis factor-α, and IL-1ß were measured. Metabonomics detection and data mining were conducted to identify the specific metabolic sponsor of the 3 proteins. The results showed that lactoferrin, α-lactalbumin, and ß-lactoglobulin protected neurons from cerebral I/R injury by increasing the level of bopindolol and subsequently inhibiting the TLR4-related pathway to different degrees; ß-lactoglobulin had the strongest activity of the 3 proteins. In summary, this study is the first to investigate and compare the protective effects of lactoferrin, α-lactalbumin, and ß-lactoglobulin in a cerebral stroke model. The results implicate TLR4 as a novel target of the 3 bioactive proteins to prevent cerebral I/R injury.

[Effect and mechanism of marrow stromal cells within cerebrospinal fluid on SOD1 transgenic mice].

OBJECTIVE: To explore the effect and mechanism of murine marrow stromal cells (MSCs) within cerebrospinal fluid (CSF) on Cu/Zn superoxide dismutase 1 (SOD1) transgenic mice. METHODS: A dose of 5×10(5) MSCs was injected into the CSF of SOD1 transgenic mice at the ages of 8, 10 and 12 weeks.Hanging wire test and motor neuron counts were used to assess disease progression of SOD1 mice. To examine the glial activation in murine lumbar spinal cord at 15 weeks of age, the sections were stained with antibody to CD11b and GFAP through immunohistochemistry.In vitro, mRNA expression of neurotrophin of MSCs was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Compared with vehicle-treated mice, intrathecally transplanted MSCs enhanced motor performance and decreased motor neuron loss. An intrathecal injection of MSCs inhibited microglial (52 ± 7 vs 38 ± 7; F = 69.3, P = 0.02) and astrocyte (79 ± 9 vs 63 ± 9; F = 40.7, P = 0.03) activity in lumbar spinal cord at the age of 15 weeks of age. MSCs cultured in an inflammatory environment expressed a higher mRNA level of neurotrophin (P < 0.05). CONCLUSION: A CSF administration of MSCs has therapeutic effects on SOD1 mice. And the meditation of MSCs may be achieved through inhibiting neuroinflammation and secreting a variety of trophic factors.

Investigation of the mechanical properties of a low-shrinkage liquid crystalline matrix combined with nano-hydroxyapatite.

The objective of this study was to investigate the mechanical properties of a low-shrinkage acrylate liquid crystalline resin matrix (ALCRM), (4-3-(acryloyloxy)-2-hydroxypropoxy) phenyl 4-(3-(acryloyloxy)-2-hydroxypropoxy) benzoate, combined with nano-hydroxyapatite (na-HA). The ALCRM monomer and diluent monomer triethylene glycol dimethacrylate (TEGDMA) were mixed at a mass ratio of 7:3 to prepare the resin matrix. The volume shrinkage of the ALCRM/TEGDMA and the traditional resin matrix Bis-GMA (2, 2-bis [4-(3-methacryloxy-2-hydroxypropoxy) phenyl] propane)/TEGDMA were measured. Then 20%, 30%, and 40% na-HA were added to ALCRM/TEGDMA and Bis-GMA/TEGDMA, respectively. Compressive strength (CS) and Vickers hardness (VHN) were tested to identify mechanical properties of the composites. The volume shrinkage of the ALCRM/TEGDMA was 3.60% ± 0.36%, which was lower than that of the traditional resin matrix. Moreover, with the same amount of na-HA filler, the mechanical properties of the ALCRM-based composites were superior to those of Bis-GMA-based composites. ALCRM-based composites exhibited the highest CS and VHN values when the filler content was 30%. These results indicate that the low-shrinkage liquid crystalline resin matrix has strong mechanical properties after incorporating the na-HA. The experimental ALCRM-based composites have promising potential for the development of novel low-shrinkage dental resins with strong mechanical properties.

[Evaluation of clinical therapeutic efficiency of immediate loaded implant].

OBJECTIVE: Clinical cases of immediate loaded implants were retrospectively analyzed, in the aim of evaluating the clinical value of immediate loading. METHODS: From July 2005 to October 2009, 99 immediate loaded implants were implanted in 29 patients. The overall data including radiography, clinical examination were collected during the follow-up periods ranged from 4 to 46 months. The implants were evaluated with the survival rate, bone resorption, soft tissue esthetics (including gingiva papilla index and pink esthetic score). RESULTS: Survival rate for immediate loaded implant was 97.0%. The average bone resorption were 0.22 mm at 4-6 months after surgery, bone increase of 0.15 mm were found at 6-12 month, and bone increase up to 0.16 mm at 12-46 months. The gingival papilla index was 2.68, while pink esthetic score was 12.58. CONCLUSION: Immediate loaded implant is an effective repairing method for patients missing teeth and the esthetics effect is ideal.