In surgical operation of the non-small cell lung cancer (NSCLC),the resection of lymph node involvement is highly correlated with the prognosis of NSCLC patients.The precise estimation of localized lymph node involved and the determination of relevant pattern of lymphadenectomy are the significant predictors for the prognosis and postoperative treatment in NSCLC patients.Therefore to identify the lymph node metastasis earlier has more important significance for tumor clinical staging and making therapeutic schedule.In recent years,the value of sentinel lymph node (SIN) has attracted more and more researchers attention in detecting the occult lymph node micrometastases and surgical staging.

Objective To explore the functions of phospholipase C (PLC)-independent protein kinase C signaling pathway (PTH/nonPLC/PKC) of parathyroid hormone (PTH) and its role in bone metabolism. Methods Osteoblasts isolated from the calvaria of 2-or 3-day-old C57BL mice, identified by alkaline phosphatase staining and Alizarin red staining, were treated for 4 h with 100 nmol/L[Gly1, Arg19]hPTH(1-28) plus 10 nmol/L RP-cAMP, 10 nmol/L[Gly1, Arg19]hPTH(1-34) plus 10 nmol/L RP-cAMP , 10 nmol/L PTH(1-34), or and 0.1% trifluoroacetic acid (TFA). The total RNA was then isolated for screening differentially expressed genes related to PTH/nonPLC/PKC pathway using Affymetrix mouse 12x135K gene expression profile microarray, and the identified genes were confirmed by real-time quantitative PCR. MC3T3-E1 cells treated with[Gly1, Arg19]hPTH(1-28)+RP-cAMP,[Gly1, Arg19]hPTH(1-34)+RP-cAMP,[Gly1, Arg19]hPTH(1-34)+RP-cAMP+100 nmol/L Go6983, or 0.1%TFA were also examined for GR(1-28)-or GR(1-34)-mediated gene expression changes using real-time quantitative PCR. Results Alizarin red staining visualized red mineralized nodules in the osteoblasts at 28 days of culture. According to the genechip results, we selected 56 target genes related to PTH/nonPLC/PKC pathway, among which CITED1 showed higher expressions in[Gly1, Arg19]hPTH(1-34)+RP-cAMP group than in both the control group and[Gly1, Arg19]hPTH(1-28)+RP-cAMP group (P<0.05), and its expression was the highest in PTH(1-34) group (P<0.05). RT-PCR of MC3T3-E1 cells yielded consist results with those in the primary osteoblasts, and the cells treated with Go6983 (a PKC inhibitor) did not show GR(1-28)- or GR(1-34)-mediated differential expression of CITED1. Conclusion The activation of PLC-independent protein kinase C signaling pathway of PTH enhances the expression of CITED1 in mouse osteoblasts to mediate the effect of PTH on bone metabolism, and this pathway is not dependent on the activation of PLC or PKA signaling.

Objective To explore the functions of phospholipase C (PLC)-independent protein kinase C signaling pathway (PTH/nonPLC/PKC) of parathyroid hormone (PTH) and its role in bone metabolism. Methods Osteoblasts isolated from the calvaria of 2-or 3-day-old C57BL mice, identified by alkaline phosphatase staining and Alizarin red staining, were treated for 4 h with 100 nmol/L[Gly1, Arg19]hPTH(1-28) plus 10 nmol/L RP-cAMP, 10 nmol/L[Gly1, Arg19]hPTH(1-34) plus 10 nmol/L RP-cAMP , 10 nmol/L PTH(1-34), or and 0.1% trifluoroacetic acid (TFA). The total RNA was then isolated for screening differentially expressed genes related to PTH/nonPLC/PKC pathway using Affymetrix mouse 12x135K gene expression profile microarray, and the identified genes were confirmed by real-time quantitative PCR. MC3T3-E1 cells treated with[Gly1, Arg19]hPTH(1-28)+RP-cAMP,[Gly1, Arg19]hPTH(1-34)+RP-cAMP,[Gly1, Arg19]hPTH(1-34)+RP-cAMP+100 nmol/L Go6983, or 0.1%TFA were also examined for GR(1-28)-or GR(1-34)-mediated gene expression changes using real-time quantitative PCR. Results Alizarin red staining visualized red mineralized nodules in the osteoblasts at 28 days of culture. According to the genechip results, we selected 56 target genes related to PTH/nonPLC/PKC pathway, among which CITED1 showed higher expressions in[Gly1, Arg19]hPTH(1-34)+RP-cAMP group than in both the control group and[Gly1, Arg19]hPTH(1-28)+RP-cAMP group (P<0.05), and its expression was the highest in PTH(1-34) group (P<0.05). RT-PCR of MC3T3-E1 cells yielded consist results with those in the primary osteoblasts, and the cells treated with Go6983 (a PKC inhibitor) did not show GR(1-28)- or GR(1-34)-mediated differential expression of CITED1. Conclusion The activation of PLC-independent protein kinase C signaling pathway of PTH enhances the expression of CITED1 in mouse osteoblasts to mediate the effect of PTH on bone metabolism, and this pathway is not dependent on the activation of PLC or PKA signaling.

OBJECTIVETo establish a model bearing human lung cancer xenograft with bone metastasis in mice with normal immune function.

METHODSForty female C57BL/6J mice were randomly allocated into 4 equal groups, including a control group and 3 immunosuppression groups treated with low, moderate, and high doses of dexamethasone (50, 100, and 150 mg, respectively). Four days after immune suppression, the mice were subjected to percutaneous injection of1.0×10(9) L(-1) A549 cells into the tibial plateau, and the bone defects were assessed radiographically 28 days after modeling. HE staining and immunohistochemical staining were used to examine the tumor tissues and bone tissue damages.

RESULTSIn each of the 4 groups one mouse died during tumor cell injection. Only 1 mouse showed tumor formation in low-dose immunosuppression group, as compared to 7 and 4 in moderate- and high-dose immunosuppression groups. X-ray and microCT scan showed significant tibial bone destruction in moderate- and high-dose groups. The moderate- and high-dose groups showed similar ALP activities but both were significantly higher than those in the other two groups (P<0.05).

CONCLUSIONImmunosuppression with a moderate dose of dexamethasone results in longer survival time of the human lung cancer xenograft-bearing model mice as well as a higher tumor formation rate.

Animals ; Bone Neoplasms ; secondary ; Cell Line, Tumor ; Dexamethasone ; pharmacology ; Disease Models, Animal ; Female ; Humans ; Immunosuppression ; Lung Neoplasms ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation

Objective To establish a model bearing human lung cancer xenograft with bone metastasis in mice with normal immune function. Methods Forty female C57BL/6J mice were randomly allocated into 4 equal groups, including a control group and 3 immunosuppression groups treated with low, moderate, and high doses of dexamethasone (50, 100, and 150 mg, respectively). Four days after immune suppression, the mice were subjected to percutaneous injection of1.0 × 109 L-1 A549 cells into the tibial plateau, and the bone defects were assessed radiographically 28 days after modeling. HE staining and immunohistochemical staining were used to examine the tumor tissues and bone tissue damages. Results In each of the 4 groups one mouse died during tumor cell injection. Only 1 mouse showed tumor formation in low-dose immunosuppression group, as compared to 7 and 4 in moderate- and high-dose immunosuppression groups. X-ray and microCT scan showed significant tibial bone destruction in moderate- and high-dose groups. The moderate- and high-dose groups showed similar ALP activities but both were significantly higher than those in the other two groups (P<0.05). Conclusion Immunosuppression with a moderate dose of dexamethasone results in longer survival time of the human lung cancer xenograft-bearing model mice as well as a higher tumor formation rate.

Objective To establish a model bearing human lung cancer xenograft with bone metastasis in mice with normal immune function. Methods Forty female C57BL/6J mice were randomly allocated into 4 equal groups, including a control group and 3 immunosuppression groups treated with low, moderate, and high doses of dexamethasone (50, 100, and 150 mg, respectively). Four days after immune suppression, the mice were subjected to percutaneous injection of1.0 × 109 L-1 A549 cells into the tibial plateau, and the bone defects were assessed radiographically 28 days after modeling. HE staining and immunohistochemical staining were used to examine the tumor tissues and bone tissue damages. Results In each of the 4 groups one mouse died during tumor cell injection. Only 1 mouse showed tumor formation in low-dose immunosuppression group, as compared to 7 and 4 in moderate- and high-dose immunosuppression groups. X-ray and microCT scan showed significant tibial bone destruction in moderate- and high-dose groups. The moderate- and high-dose groups showed similar ALP activities but both were significantly higher than those in the other two groups (P<0.05). Conclusion Immunosuppression with a moderate dose of dexamethasone results in longer survival time of the human lung cancer xenograft-bearing model mice as well as a higher tumor formation rate.