Inactivation of the Rho-ROCK signaling pathway to promote neurologic recovery after spinal cord injuries in rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>After injury, axonal regeneration of the adult central nervous system (CNS) is inhibited by myelin-derived growth-suppressing proteins. These axonal growth inhibitory proteins are mediated via activation of Rho, a small GTP-binding protein. The activated form of Rho, which is bound to GTP, is the direct activator of Rho kinase (ROCK) through serial downstream effector proteins to inhibit axonal regeneration. The objective of this study was to observe the therapeutic effect of inactivation of the Rho-ROCK signaling pathway to promote neurologic recovery after spinal cord injuries in rats.</p><p><b>METHODS</b>One hundred and twenty adult female Sprague-Dawley rats were randomly divided into three groups. Laminectomies alone were conducted in 40 rats in the sham group. Laminectomies and spinal cord transections were performed in 40 rats in the control group (treated with normal saline administered intraperitoneally). Laminectomies and spinal cord transections were performed in 40 rats in the fasudil-treated group (treated with fasudil administered intraperitoneally). Neurologic recovery was evaluated before surgery and 3 days, and 1, 2, 3, and 4 weeks after surgery using the Basso-Beattie-Bresnahan (BBB) scale of hind limb movement. At the same time, the expression of RhoA mRNA was determined with RT-PCR. Histopathologic examinations and immunofluorescence staining of NF were performed 1 month after surgery.</p><p><b>RESULTS</b>Compared with the control group, the BBB scores of the fasudil-treated group were significantly increased and the expression of RhoA mRNA was significantly decreased. In the fasudil-treated group, a large number of NF-positive regenerating fibers was observed; some fibers crossed the slit of the lesion.</p><p><b>CONCLUSION</b>Inactivation of the Rho-ROCK signaling pathway promotes CNS axonal regeneration and neurologic recovery after spinal cord injuries in rats.</p>

Integrated assessment of potential value of erbB-2 amplification or expression status as novel therapy target in Chinese children and adolescents with osteosarcoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Targeted tumor therapies have been making rapid progress in recent years, and the erbB-2 oncogene is a suitable target. There was much discussion about the level of erbB-2 in osteosarcoma. The aim of this study was to investigate the erbB-2 amplification or expression status in osteosarcoma.</p><p><b>METHODS</b>Fluorescence in situ hybridization (FISH) and DNA probes for erbB-2 and centromere 17 were used to examine the erbB-2 gene amplification status in 32 osteosarcoma samples, and expression of erbB-2 was analyzed by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>None of the 32 osteosarcomas was observed by FISH to have the erbB-2 gene amplified, and no distinguishable membrane staining was seen in any case yet, nevertheless, erbB-2 overexpression was present in 6 tumor samples by RT-PCR.</p><p><b>CONCLUSIONS</b>The status of erbB-2 gene amplification and membrane overexpression is rare in osteosarcomas, and might suggest that the erbB-2 target agent should not be applied to osteosarcomas as single treatment.</p>

The in vitro differentiation and the variant expression of protein of bone marrow stromal stem cells when treating the spinal cord injury / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the differentiation and the variant expression of protein of the bone marrow stromal stem cells (BMSCs) when the BMSCs differentiated into the neuronal cells in the analogous micro-environment of spinal cord injury.</p><p><b>METHODS</b>BMSCs were isolated from bone marrow of Wistar rats and labeled with PKH26 (control group), and then were cocultured with neural cells, which were isolated from the spinal cord of the fetal rats, in the same plate well (co-culture group) or in the two-layer Petri well (two-layer group). Eight days later, the BMSCs were identified by immunofluorescence staining of NSE and GFAP respectively. The apparently changing proteins were analyzed by SELDI-TOF-MS while the BMSCs differentiated into neurons.</p><p><b>RESULTS</b>Eight days after co-culturing with neural cells in the same plate well or in the two-layer Petri well, BMSCs appeared more similar with neural cells. The immunofluorescence identification showed that, NSE and GFAP of which the BMSCs of the two-layer group expressed were obviously higher than control group (P < 0.05); and these two proteins of co-culture group were also obviously higher than the other two groups (P < 0.05). Five proteins in the co-culture group changed obviously as followed: TIP39_RAT and CALC_RAT were 5.360 and 2.807 times of that in the control group; INSL6_RAT, PNOC_RAT and PCSK1_RAT were 38.0, 49.9 and 43.8 percent of those in the control group.</p><p><b>CONCLUSIONS</b>BMSCs could differentiate into neural cells in vitro, and the differentiation ratio of BMSCs in the co-culture group is higher than that of the two-layer group. Five proteins, including TIP39_RAT, CALC_RAT, INSL6_RAT, PNOC_RAT and PCSK1_RAT, are correlated closely to the mechanisms of which the BMSCs differentiated into neurons.</p>

Study on inhibition of acute rejection in rat limb allografts by inducible costimulator pathway blockade / 中国地方病学杂志

Objective To observe the effect of inducible costimulator(ICOS) costimulation pathway blockade in rat limb allografts acute rejection by RNA interference. Methods Twenty-seven cases of modified hind llmb allotransplantation were performed from Wistar to SD rats. The rats were divided into 3 gronps(each n=9): the rejection group not given a special disposal; the control group, consisting of SD rats that received injection of pSilencer 4.1 and Sofast complex by vein post transplantation; and the interference group that received injection of pSilencer 4.1-ICOSshRNA and Sofast complex. On the eighth day posttransplantation, 3 rats were killed to study the pathological changes in each group. The expressions of ICOS gene in vivo were detected by flow cytometry and RT-PCR. The mixed lymphocyte reaction (MLR) was performed and eytokines in blood were measured by ELISA. The rest rats were used to record limb survival time. Results The mean survival time in rats of the rejection and the control groups were(11.34±1.21) and (11.14±1.32) days respectively. In the interference group, the mean survival time of limb allografts was (16.85±1.73) days(P<0.05). The rats in the rejection and the control groups experienced moderate to serious acute rejections with skin epidermal necrosis, a large quantity of lymphocyte infdtration, muscle cell necrosis and interstitial edema, while the pathological changes in rats of the interference group were mild. The splenocyte ICOS mRNA expression level in the interference group(18.75%) was significantly lower than that of the rejection group(100%) and the control group(98.51%). ICOS cell surface expression level as judged by the fluorescence intensity was 45.59±12.87 in the interference group, 103.72±21.76 in the rejection group, and 93.47±29.55 in the control group(F=6.89, P<0.05). In stimulation assays, a one-way mixed lymphocyte reaction stimulation index(SI), with spleen cells from Wistar and Lewis rats, respectively, the rejection group (5.26±0.42,5.18±0.29) and the control group (5.37±0.27,4.93±0.44) had significantly greater reactions than the interference group(2.37±0.35, 4.87±0.36), respectivily(F=7.29, P<0.05; F=6.19, P0.05). In the IFN-γ and IL-4 expression assays, reactions of the interference group (230.17±38.47,160.32±59.13) were lower than those of the rejection group(490.73±51.48,230.67±45.21) and the control group(480.15±43.96, 240.53± 47.36), (F=7.23,6.75, all P<0.01). Conclusions In vivo transfection of pSilencer 4.1-ICOS shRNA interference plasmid can effectively block T-cell co-stimulation pathway, suppress acute rejection, and prolong limb allografts survival.

Study of reduction rat T-lymphocytes activation and proliferation by inhibition inducible co-stimulator gene expression / 中华外科杂志

<p><b>OBJECTIVE</b>To observe the change of activation and proliferation ability of rat T-lymphocytes after suppress ICOS gene expression by RNA interference.</p><p><b>METHODS</b>Four interference sites targeting at rat ICOS gene were designed and four pairs of oligonucleotide fragments were cloned into the pSilencer 4.1-CMV neo plasmid vectors then transfected into rat lymphocytes with cationic liposome. The expression of mRNA and protein of ICOS was detected by RT-PCR and flow cytometry. The alteration of lymphocyte proliferation ability was evaluated by mix lymphocyte reaction, and the secretion levels of IFN-gamma and IL-4 were measured by ELISA procedure.</p><p><b>RESULTS</b>After transfection, the expression of mRNA and protein of ICOS in test groups were lower than that in control groups (P < 0.05). The ability of T-lymphocytes in proliferation was poor and the levels of IFN-gamma and IL-4 were reduced with ICOS gene shut down.</p><p><b>CONCLUSIONS</b>RNA interference plasmid vector can suppress ICOS expression in rat T-lymphocytes significantly, and it may be useful for further study on transplantation immunity tolerance.</p>

Reconstruction of large limb bone defects with a double-barrel free vascularized fibular graft / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The use of a free, vascularized fibular graft is an important technique for the reconstruction of large defects in long bones. The technique has many advantages in strong, tubular bones; a more reliable vascular anatomy with a large vascular diameter and long pedicle is used, minimizing donor-site morbidity. Due to limitations in both fibular anatomy and mechanics, they cannot effectively be used to treat large limb bone defects due to their volume and strength.</p><p><b>METHODS</b>From 1990 to 2001, 16 clinical cases of large bone defects were treated using vascularized double-barrel fibular grafts. Patients were evaluated for an average of 10 months after surgery.</p><p><b>RESULTS</b>All the patients achieved bony union; the average bone union took 10 months post surgery, and no stress fractures occurred. Compared with single fibular grafts, the vascularized double-barrel fibular grafts greatly facilitate bony union and are associated with fewer complications, suggesting that the vascularized double-barrel fibular graft is a valuable procedure for the correction of large bone defects in large, long bones in addition to enhancing bone intensity.</p><p><b>CONCLUSIONS</b>The vascularized double-barrel fibular graft is superior to the single fibular graft in stimulating osteogenous activity and biological mechanics for the correction of very large bone defects in large, long bones. Free vascularized folded double-barrel fibular grafts can not only fill up large bone defects, but also improve the intensity margin. Therefore, this study also widens its application and enlarges the treatment targets. However, in the case of bone deformability, special attention should be paid to bone fixation and protection of donor and recipient sites.</p>

A 4-year follow-up of functional rehabilitation in a patient with allografted forearms / 中华创伤骨科杂志

Objective To report the functional reconstruction and rehabilitation for a patient who under- went allograft for both of his forearms and hands.Methods One male patient underwent allograft for both of his forearms and hands in October 2002 in our department to reconstruct his hand functions.The allografted hands were intervened with an integrated rehabilitation program,which involved administration of immunosuppressants,post- operative monitoring,postoperative functional training,massage,physiotherapy,orthosis,performance training, sensation training,secondary operation and mental rehabilitation.The patient was followed up for 4 years.Results The forearms and hands of the patient were in good shape and regained nearly normal sensation.The distance of two-point-discrimination was 2.5 cm to 4.0cm.The TAM (total active motion) of fingers was fine.The patient could look after himself well and were healthy in psychology.Conclusion An integrated rehabilitation program can yield satisfactory results in the management of allografted forearms and hands.