Constructing CDNA Subtracted Libraries And Screening The Abnormal Expression Of Genes In Labial Glands From Sj(?)gren's Syndrome Patients And Study Their Functions

ObjectivesTo constructing two cDNA subtracted libraries and screen the abnormal expression of gene in labial glands from Sjogren's syndrome(SS) Patients. Then some genes that may be associated with SS were selected for studying pathogenesis at level of mRNA and protein.MaterialsThe biopsy specimens of labial gland from Sjogren's syndrome and control patients were collected in the stomatological clinic of Peking Union Medical College Hospital from September in 2004 to April in 2006. The subjects were divided into two groups: One was the primary Sjogren's syndrome(pSS) that was calibrated by the American-European consensus criteria for SS(2002) and excluded those with other connective diseases. The other group acted as controls. Some had only complaint of eye-dry and/or mouth-dry, the objective tests such as salivary flow rate, Shirmer test, autoimmuoantibodies and labial gland biopsy study were normal. Others were labial trauma or lip cyst without systemic disease. The difference of age between two groups was not significant tested by statistics.Informed consent was obtained from all tissue donors and information of patients and results of clinical tests was obtained from clinical record.Methods1. Constructing the subtracted cDNA library: Total RNA was isolated with TRIR (Total RNA Isolation Reagent, ABgene) from biopsy specimens of labial glands. Then subtractive hybridization using Clontech PCR-Select^TM cDNA Subtraction Kit, (Clontech) enabled us to compare two populations of mRNA and obtained two subtracted libraries. The different expressed clones were directly inserted into an A/T cloning vector to construct forward and reverse cDNA libraries.2. Selecting different expressed genes: 384 clones randomly selected from forward and reverse libraries respectively hybrided with probes, the second PCR products marked with [α-³²P]-dATP. The clone that signal ratio was more than 2 or less than 0.5 was sequenced by primers SP6 and T7 on ABI 377 sequencor (Perkin-Elmer). The sequences were blasted on www.ncbi.nlm.nih.gov and obtained Genebank ID and gene names. After reviewing literature, some genes associated with SS were selected as target genes for further studying.3. Semi-quantitative analyzing mRNA of target genes: After specific primer synthesized based on the sequences of target genes and house-keeping geneβ-actin, the target genes andβ-actin acted as templates to perform reverse transcript polymerase chain reaction (PCR). The PCR products showed bands on gel after electrophoresis, Then mRNA expression of target genes were semi-quantitated by the OD ratio of the target genes toβ-actin bands under densitometry.4. Immunohistochemistry: Neutral-buffered formaldehyde fixed, paraffin-embedded tissue sections (4 um) were mounted on slides. The slides were incubated with diluted MUC5B(1:5 diluted) polyclonal antibody (Santa Cruz) and MUC5B-positive acini were shown brown under microscopy. Sum of intensity of brown stain in insights was calculated automatically by compute with the Image-ProPlus 5.0. The sum of intensity indicated the quantity of protein.5. Statistics analyzing: After constructing database by Excel, Comparing between two groups and correlation study was used the software of Statistics Pakage for Social Science (PSS11.0). When P is less than 0.05, the statistics significance is confirmed.Results1. There was not significant different between the age of two groups by t test. The age was comparable in two groups.2. 99 out of 768 randomly selected clones were different expressed confirmed by Northern blot, which ratio was more than 2 or less than 0.5.63 clones were sequenced successfully and blasted on internet. The coincidence of base pair was over 98%. The name and GeneBank ID of these clones were obtained.3. MUC5B and MUC7 were found lower expressed in SS group than controls after semi-quantitative PCR. Statistics test was done, t was 2.4,2.3 respectively and P was 0.032, 0.021 respectively.4. The mRNA transcription of both MUC5B and MUC7 in labial glands from Sjogren's syndrome patients were negatively correlated with the lymphocyte focus score (LFS) and the titer of anti-SSA antibody. The coefficient of MUC5B between the factors was-0.387 and-0.474 respectively, the P was 0.032 and 0.013 respectively; The coefficient of MUC7 between the factors was-0.386 and-0.595 respectively, the P was 0.032 and 0.001 respectively. Multiple regression study indicated the two mRNAs only associated with the titer of anti-SSA antibody, the coefficient of MUC5B and MUC7 was 0.714 and 0.708 respectively, the both P were 0.001.5. The protein of MUC5B mainly distributed on the labial gland acini, the ducts were less staining. On the sections from Sjogren's syndrome patient, MUC5B was decreased statistically (P＜0.05) comparing with that in controls.6. The protein of MUC5B in labial glands from sjogren's syndrome patients was negatively correlated with LFS and the titer of anti-SSA antibody and was positively correlated with mRNA. The coefficient of MUC5B between 3 factors was-0.396, -0.546 and 0.750 respectively, the P was 0.041, 0.003 and 0.000 respectively. Multiple regression study indicated the only mRNA associated with protein of MUC5B, the coefficient was 113.664, the P was 0.000.Conclusions1. Supression subtractive hybridization is a powerful technique to compare two population of mRNA and obtain clones of genes that are expressed differently. This technique can be used for studying pathogenesis of autoimmune disease.2. The subtractive cDNA library of labial glands of SS was constructed successfully. There is a great number of genes different expressed in SS. Some clones screening from cDNA library may be the virulence gene of SS and it is necessary to study further more.3. The mRNA of MUC5B and MUC7 were lower expressed in labial glands of SS than that controls. The mRNAs lowing down might be take place at the early stage of disease and they were not associated with all clinical index but with the titer of anti-SSA antibody, it suggested that autoimmnoantigodies might play an important role.4. The MUC5B and MUC7 changed on common at the level of mRNA in course of disease and they had the same affect factors. It was indicated that the two gene expressing lowly on the same principle.5. The protein of MUC5B in labial glands from Sjogren's syndrome patients was negatively correlated with LFS and the titer of anti-SSA antibody and was positively correlated with mRNA. Multiple regression study indicated the only mRNA associated with protein of MUC5B, these suggested that MUC5B mRNA was the immediate cause of the protein lowing down.