BACKGROUND: The existence of chronic heel pain induced by the injury of inferior calcaneal nerves prompted us .to conduct an anatomic study of the innervations of the medial and inferior calcaneal nerves, so as to avoid nerve injury during plantar operation.OBJECTIVE: To study the innervations and course of the medial and inferior calcaneal nervesDESIGN: Single specimen experiment.SETTING: Anatomical Laboratory of Shenyang Medical College.PARTICIPANTS: This study was completed at the Anatomical Laboratory of Shenyang Medical College between August 2002 and December 2004.Totally 36 lower limbs from volunteer donators or adult corpus provided by related department were routinely antisepticised for anatomical study.METHODS: Tibia nerve was separated from the middle 1/3 of leg until it branches into the medial plantar nerve (MPN) and the lateral plantar nerve (LPN), so as to investigate the origin, course, branches and innervation of the medial and inferior calcaneal nerve (MCN and ICN). The line linked the medial condyle center and plantar center was used as reference, nerve scored"0"if passed through the line, and scored positive value if above and negative value if below the line.MAIN OUTCOME MEASURES: The origin, course, branches and innervations of MCN and ICN in adults.RESULTS: Both medial and inferior calcaneal nerve could be identified in all 36 specimens. ① The source of MCN: 27 (75%) were found deriving from tibia nerve, 7 from LPN (19%) and 2 from tibia nerve and LPN (6%).② The branches of medial plantar nerve: 29 (81%) nerves had major branches, most of which lay superficial to the abductor hallucis muscle (AH) in the fat pad or under skin. ③ The source of ICN: 31 (86%) were found deriving from LPN, 3 from tibia nerve (8%) and 2 from MPN (6%).④ The origin of inferior calcaneal nerve: At (1.7±4.5) cm beneath the reference. ⑤ The branch and innervations of ICN: 24 nerves had two major branches (67%), most of which lay superficial to the medial edge of plantar quadratus.CONCLUSION: The origin, course, and innervations of MCN and ICN were relatively stable, it is preferable to take lateral process in plantar operation, and should be as rear as possible if taking inferior process so as to avoid nerve injury.

Objective To construct miR-223 knockdown lentivirus vector and provide a tool for further study of the function of miR-223. Methods According to the Invitrogen miR-RNAi online design tool, a pair of complementary oligo?nucleotides encoding miR-223 mature sequence was designed, annealed and ligated with pcDNA6.2-GW/EmGFP-miR vec?tor. Then miR-RNAi expression cassette was cut and subcloned into lentiviral pCDH-CMV-MCS-EF1-copGFP vector. The lentiviruses were packaged and titered, and then ST2 cells were infected with viruses. The efficiency of infection was calcu?lated, and the knockdown of endogenous miR-223 was detected by using real-time RT-PCR. Results Restriction enzyme digestion and sequencing results showed that miR-223 lentivirus construct was successfully made. Lentivirus that knock?down miR-223 expression packaged and infected of target cells. The expression of GFP green fluorescent protein accounted for 80%-90%and the virus titer was 1×109 PFU/mL. The infection efficiency reached 90%. Compared with negative control virus, miR-223 knockdown lentivirus significantly down-regulated the expression of miR-223 in ST2, and was 31%(n=3, t=15.091, P<0.05). Conclusion miR-223 knockdown lentivirus is successfully made. It provides a tool for further studying the function of miR-223.

Objective To investigate the effects of the traditional herb-epimedium on the expression of interleukin-6 (IL-6) mRNA in bone of ovariectomized rat. Methods Forty female rats were randomly allocated into 4 groups, 10 in each: ovariectomized (OVX) group, sham operation group, OVX followed by epimedium (group 3)or nilestriol (group 4) for 3 months respectively. All rats were then sacrificed,and total RNA were directly isolated from their right tibia. Interleukin-6 mRNA expression was detected by relative semiquantitative reverse transcription-polymerase chain reaction technique. Lumbar bone mineral density (BMD) was measured by dual-energy X ray absorptiometry before sacrifice. Results The BMD of epimedium group was significantly higher than that in the OVX group ( P

Objective To investigate the regulation of parathyroid hormone(1-34) on mRNA expression of osteoclast inhibitory lectin (OCIL) gene in UMR106 osteoblastic-like cells and involved signaling pathway.Methods Rat UMR106 osteoblastic-like cells were cultured and treated with various concentration of PTH(1-34) and specific agonists or inhibitors of PKA,PKC,Ca2+/calmodulin-dependent protein kinase (CaMK) and mitogen-activated protein kinase (MAPK) signal pathways for indicated time intervals.Then the cells were gathered at indicated time points and total RNA were extracted.OCIL mRNA expression was analyzed using real-time PCR technique.Results PTH(1-34) stimulated OCIL mRNA expression in a time-and dose-dependentmanner.A dose of 10 nmol/L PTH(1-34) started to induce OCIL mRNA from 6 h,with a highest increase of about 2.8-fold vs.control group (without PTH treatment) at 24 h.The up-regulation of OCIL mRNA began and reached maximum later than RANKL induction and OPG suppression effected by PTH(1-34).Protein Kinase A (PKA) signaling activators forskolin(FSK) and dibutyryl cAMP (db-cAMP),as well as calcium ionophore A23187 all up-regulated OCIL mRNA with the maximal induction of about 4.2-fold,4.5-fold and 5.1-fold.Protein Kinase C (PKC) activator phorbol-12-myristate-13-acetate(PMA) reduced OCIL mRNA expression at the early stage(2-6 h),with the highest down-regulation of 50％ at 6 h.However,the inhibitory effect on OCIL mRNA turned into slightly stimulatory effect later (24 h).PKA inhibitor KT5720,calmodulin antagonist W-7,CaMK Ⅱ inhibitor KN-62 and mitogen-activated protein kinase (MAPK) inhibitor PD98059 all blocked PTH(1-34)-induced OCIL mRNA expression by the maximal reduction of 56％,61％,63％ and 50％ respectively.There also exist cross-talks between different signal pathways.MAPK inhibitor PD98059 blocked the expression of OCIL mRNA which was stimulated by PKA activators FSK or db-cAMP,with the reduction of 98％ and 63％ respectively,while the OCIL mRNA expression stimulated by A23187 remained unaffected.Conclusion PTH(1-34) increased OCIL mRNA expression in vitro through cAMP/PKA,Ca2+/CaMK and MAPK signaling pathways.

Background and purpose:Gene therapy is a novel approach for the treatment of the patients with breast cancer. One of the effective ways is to direct transgenic expression to specific tissues or tumors with the use of tissue-specific-promoters (TSP). hTERT (human telomerase reverse transcriptase) is highly expressed in many types of cancers including breast cancer. Thus, we hypothesized that the hTERT promoter targeting with gene therapy vectors could be exploited for breast cancer. In this study, we amplified hTERT gene promoter and cloned it into the reporter vector pEGFP and pGL3-Basic. Afterwards, the specific transcription of hTERT promoter in MCF7 cells was evaluated. Methods:hTERT gene minimal promoter was PCR amplified and cloned into the reporter plasmid pEGFP-1 and pGL3-Basic.The constructs pEGFP/TERT and pGL3/TERT were transfected into MCF7 breast cancer cells and HBL100 human epithelial cells, respectively.The expression of EGFP and luciferase were investigated, respectively..Results:pEGFP/TERT and pGL3 /TERT bearing hTERT gene promoter were constructed. The specific expression of EGFP was detected in MCF7 cells while little expression of EGFP was seen in HBL100 cells.In accordance with EGFP, luciferase driven by hTERT also showed specific and high activity in MCF7 cell (RLU/U: 33784), which is 15 times higher than in HBL100 (RLU/U: 2400).Conclusions:The high transcriptional activity of hTERT gene promoter in MCF7 cell indicates its potential utility as a novel candidate for transcriptional targeting of breast cancer.

Objective:To identify the regulation of monoeyte chemoattractant protein-1(MCP-1)in osteoblasts by parathyroid hormone related protein(PTHrP) in breast cancer cells.Methods:Osteoblast-like UMR106 cells were treated with 10~(-8)mol/L PTHrP (1-34) for different times and the expression of MCP-1 was studied by semiquantitative RT-PCR.Recombinant shRNA plsmid PTH1R/pRNAT was constructed and transfected into UMR106 cells.Stable PTH1R/UMR106 cells were established,in which PTH1R expression was knocked-down.The conditioned medium(CM) of breast cancer MDA-MB231 was added to UMR 106 and PTH1R/UMR106 cultures respectively.Thereafter,mRNA of MCP-1 was examined by semiquantitative RT-PCR.Results:PTHrP induced expression of MCP-1 in UMR106 cells and the induction began at 3 h and reached the maximum at 6 h.Treatment of UMR106 cells with CM of MDA-MB231 also significantly induced mRNA of MCP-1 at 6 h(1.238 1±o.115 5 and 0.598 4±0.036 4,P＜0.05).This effect was partly blocked by the knockdown of PTH1R in UMR106 cells(0.867 2±0.045 7,P＜0.05).Conclusion:The osteoblastic MCP-1 is up-regulated in breast cancer through expression of PTHrP.

Sodium phenylacetate can induce differentiation of tumor cells and has been approved as a drug for the treatment of adults with cancer. In order to explore the immunological mechanism of its antitumor effect, the influence of sodium phenylacetate on HLA class I and II molecule expression in various human tumor cell lines, including breast adenocarcinoma (MCF-7.MUA-453), gastrocarcinoma(MKN-28,MKN-45), ovarian cancer(3AO) and cervical cancer(Hela), was studied with ELISA. The result showed that HLA class II molecule was absent from the surface of MCF-7 cells, but they could be induced after 7 days of continued treatment with sodium phenylacetate. Sodium phenylacetate was found to increase HLA class I molecule expression on the surface of MCF-7 cells, HLA class 1 and II molecule expression on the surface of MDA-453、 MKN-28、 MKN-45、 Hela and 3AO cells. The effect of sodium phenylacetate on HLA class I molecule expression in tumor cells is dose-and time-dependent.

A crucial role in eliciting anti-tumor immunity of costimulatory molecule CD80(B7-1) has been demonstrated by animal experimental studies.In this paper, CD80 expression in human tumor cell lines and EBV-transformed B cell was detected using RT-PCR and FACS methods. The results showed that CD80 expression of Raji and EBV-transformed B cell was positive but that of 3AO,MCF-7,MDA-453,MKN-45, Hela was negative. We have constructed retroviral vector CD80-pLN,transfected package cell PA317, screened high titer rctrovirus supernatant then infected human tumor cell lines and gained CD80 positive tumor cell clones. With pre-and post-CD80-transfected human breast carcinoma cell line MDA-453,the upregulated expression of ICAM-I, HLA class I molecule was observed, but the expression of HLA class II molecule wasn't changed.

To study the induced condition and characteristics of T cell anergy in vitro. Methods: Anergic Tcell was induced by combination of B7-1 mAb and cyclosporin A (CsA) in vitro, cytokine gene of anergic T cells was detected by RT-PCR. Results: T cell anergy was antigen-specific. The state of T cell anergy can be reversed by PHA, CD3 mAb and PMA plus A23187. IL-2 can prevent the induction of T cell anergy, but it can not reverse the state of un-responsiveness. IL-2 and IFN mRNA can not express in anergic T cells. In contrast, IL-4 and IL-10 mRNA were detectable. Conclusion: T cell anergy can be induced in vitro , cytokine profile of anergic T cells deviated to Th2-like phe-norype.

Objective To construct a luciferase reporter vector containing the 3′untranslated region (3′UTR) of nuclear factor I-C (nuclear factor I-C, Nfic), and apply dual luciferase reporter gene system to determine the association between microRNA-20a (miR-20a) and its potential target gene Nfic. Methods The potential complementary binding sites of miR-20a and Nfic were predicted by Targetscan. The 3′UTR of Nfic fragment amplified by PCR was cloned into luciferase reporter vector MIR- Report Luciferase. The luciferase reporters containing 3′ UTR of Nfic and miR- 20 mimics (experimental group) or NC mimics (control group) were co-transfected into 293-AD cells. Cells were collected, and then dual-luciferase reporter assay was performed to detect the luciferase activity of the two groups of cells, consequently the relationship between miR-20a and Nfic was identified. The miR-20a mimics and NC mimics were transfected into marrow stromal cell line ST2 respectively. The total cell lysates were collected, and the expression level of NFIC was detected by Western blotting assay. Results Results of double enzyme digestion and DNA sequencing showed that sequence of luciferase reporter vector was correct. miR-20a specificity bounded to Nfic 3′UTR and inhibited the luciferase activity of the reporter construct (P<0.05). Western blotting assay showed that the NFIC protein level was obviously down-regulated in ST2 cells after the transfection of miR-20a mimics compared with that of control. Conclusion The luciferase reporter vector containing the 3′UTR of Nfic is constructed successfully, which confirms that miR-20a can direct effect on Nfic3′UTR and repress its luciferase activity.