A Comparative Study on the Incidence, Aggravation, and Remission of Lupus Nephritis Based on iTRAQ Technology.

AIM AND OBJECTIVE: Lupus nephritis (LN) is one of the major complications of systemic lupus erythematosus (SLE). The specific mechanisms of pathogenesis, aggravation, and remission processes in LN have not been clarified but is of great need in the clinic. Using isobaric tags for relative and absolute quantitation (iTRAQ) technology to screen the functional proteins of LN in mice. Especially under intervention factors of lipopolysaccharide (LPS) and dexamethasone. METHODS: Mrl-lps mice were intervened with LPS, dexamethasone, and normal saline (NS) using intraperitoneal injection, and c57 mice intervened with NS as control. The anti-ANA antibody enzyme-linked immunosorbent assay (ELISA) was used to verify disease severity. Kidney tissue is collected and processed for iTRAQ to screen out functional proteins closely related to the onset and development of LN. Western blot method and rt-PCR (real-time Polymerase Chain Reaction) were used for verification. RESULTS: We identified 136 proteins that marked quantitative information. Among them, Hp, Igkv8-27, Itgb2, Got2, and Pcx proteins showed significant abnormal manifestations. CONCLUSION: Using iTRAQ methods, the functional proteins Hp, Igkv8-27, Itgb2, Got2, and Pcx were screened out for a close relationship with the pathogenesis and development of LN, which is worth further study.

De-differentiation response of cultured astrocytes to injury induced by scratch or conditioned culture medium of scratch-insulted astrocytes.

Our previous reports indicated that astrocytes (ASTs) in injured adult rat spinal cord underwent a process of de-differentiation, and may acquire the potential of neural stem cells (NSCs). However, the AST de-differentiation and transitional rejuvenation process following injury is still largely unclear. The aim of the present study was to determine whether injured in vitro ASTs can re-enter the multipotential-like stem cell pool and regain NSC characteristics, and to further understand the mechanism of AST de-differentiation. We used an in vitro scratch-wound model to evoke astrocytic response to mechanical injury. GFAP and nestin double-labeled indirect immunofluorescence were carried out to characterize these scratched cells at various periods. Western-blot analysis was used to determine the changes of GFAP and nestin expression following injury. Furthermore, the rate of proliferation was determined by immunocytochemical detection of BrdU incorporating cells. These scratch-wound ASTs were cultured with stem cells medium to explore their ability to generate neurospheres and examine the self-renewal and multi-potency of such neurospheres. Moreover, scratched AST culture supernatant as conditioned cultured medium (ACM) was used to investigate if some diffusible factors derived from injured ASTs could induce de-differentiation of AST. The results showed: (1) the nestin positivity first appeared in GFAP-positive cells at the edge of the scratch, subsequently, disseminated into un-insulted zone. The expression of nestin in AST was increased with longer culture, while that of GFAP was decreased. Furthermore, these nestin-immunoreactive ASTs could generate neurospheres, which showed self-renewal and could be differentiated into neurons, ASTs and oligodendrocytes. (2) Scratched ASTs culture supernatant can induce astrocytic proliferation and de-differentiation. These results reveal that the in vitro injured ASTs can de-differentiate into nestin-positive stem/precursor cells, the process of de-differentiation may arise from direct injury or some diffusible factors released from injured ASTs.

[Preparation and characterization of monoclonal antibodies against rat Nogo molecule].

AIM: To prepare monoclonal antibodies against rat Nogo molecule and study the distribution of Nogo in rat central nervous system (CNS). METHODS: Murine mAbs were prepared by hybridoma technique. The distribution of Nogo in rat CNS was identified by immunofluorescent histochemical staining. RESULTS: Three hybridoma cell lines, FMU-Nogo1, FMU-Nogo2 and FMU-Nogo3, secreting mAbs against rat Nogo molecule were obtained. The titers of ascitic mAbs reached to 10(-6) and the Ig subclass of FMU-Nogo1, FMU-Nogo2 and FMU-Nogo3 was IgG2b(kappa), IgG1(kappa) and IgG1(kappa), respectively. Moreover, these mAbs could be used in immunofluoresent histochemical staining. The results showed that rat Nogo molecule could be detected in Purkinje cell layer and granual layer of rat cerebellum and on oligodendrocyte in white matter of rat spinal cord.Furthermore, these mAbs could be used in Western blot for detecting Nogo molecule present in rat spinal cord. CONCLUSION: Three mAbs against rat Nogo molecule were successfully prepared, which can provide a useful tool in research on the structure and functions of Nogo molecule in CNS.

Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain.

Phenotypic modification of dorsal root ganglion (DRG) neurons represents an important mechanism underlying neuropathic pain. However, the nerve injury-induced molecular changes are not fully identified. To determine the molecular alterations in a broader way, we have carried out cDNA array on the genes mainly made from the cDNA libraries of lumbar DRGs of normal rats and of rats 14 days after peripheral axotomy. Of the 7,523 examined genes and expressed sequence tags (ESTs), the expression of 122 genes and 51 expressed sequence tags is strongly changed. These genes encompass a large number of members of distinct families, including neuropeptides, receptors, ion channels, signal transduction molecules, synaptic vesicle proteins, and others. Of particular interest is the up-regulation of gamma-aminobutyric acid(A) receptor alpha5 subunit, peripheral benzodiazepine receptor, nicotinic acetylcholine receptor alpha7 subunit, P2Y1 purinoceptor, Na(+) channel beta2 subunit, and L-type Ca(2+) channel alpha2delta-1 subunit. Our findings therefore reveal dynamic and complex changes in molecular diversity among DRG neurons after axotomy. Sequences reported in this paper have been deposited in the GenBank database (accession numbers BG 662484-BG 673712)