Effects Of Xianhuayin On The Proliferation In Culturd Human Epithelial Cells And Expressions Of PCNA In Huma Oral Lichen Planus

Objective: The oral lichen planus (OLP) is a common oral mucosa disease and can not be cured entirely. Two to three percent of OLP patients may occur canceration. Its etiology is unknown. Multiple factors such as heredity, environment, life style and so on may cause it. The epithelial atrophic change of OLP has close relation with chronic protraction course of disease and region canceration. OLP is listed as precancerous state by WHO. At present, there is no specific treatment can cure it .Using western medicine causes adverse reaction usually. In recent years, treatment of traditional Chinese medicine shows satisfactory clinical effect.When the balance between apoptosis and proliferation is broken, malignant tumor would occur. How to discover the high risk of precancerous change in time and find a reliable method to guide the clinical therapy and evaluate the risk of the disease has become an important subject in oral clinical domain .Proliferating cell nuclear antigen (PCNA) is a 36KD neucleoprotein, an accessory protein of DNA pclymerase and has been found as a very important factor to switch on cell proliferation. Having been confirmed, PCNA increased on G1 period, achieved summit on S period and dropped the lowest on M period. PCNA has been found as independent index on bronchogenic carcinoma, carcinoma of bladder and carcinoma of stomach. Following the investigation and cognition to PCNA, we can assess the cell proliferating condition with it.We applied Xianhuayin to cure oral lichen planus and got satisfactory clinical effect at prophase clinical research. We adopted combined method of DispaseⅡand Trypsin to culture oral epithelial cells. We added Xianhuayin to culture system and observed its effect to oral epithelial cells. Many scholars had been devoting themselves to obtain consummate culture system of oral epithelial cells .But it was difficult because of microbiotic pollution,limit of source of human mucosa,difficulty adherence and multiplicity nutritional ingredient of oral epithelial cells. We analyzed above factors and improved experiment methods to explore the better culture style of epithelial cells. This paper investigated the expression of PCNA in progression of OLP.Method:1 oral epithelial cells culturePrimary culture: Specimens were obtained from healthy humans undergoing surgery for impacted third molar and mucocele removal .The specimens were sended to superclean bench in half an hour. Them were degermed by Benzalkonium bromide and gentamicin sulfate and then trimmed of excess submucosal connective tissue and digested in 0.25 % DispaseⅡ(Gibco, USA) at 4℃for 16～20h .Surface epithelium was stripped from submucosal connective tissue and treated with a trypsinization mixture(1:1mixture of 0.25% trypsin and 0.02% EDTA) for 5-8 minutes to dissociate the cells .10% fetal bovine of PBS was then added to inactivate the trypsin and EDTA. The cells were resuspended in Keratinocyte-SFM (Gibco USA), counted and seeded at approximately 2X105 viable cells/cm2 into plastic flask (NUNC, Denmark). The medium was changed every 2 days. Incubation was carried out in a humidified environment containing 5% carbon dioxide at 37℃. When primary cultures reached 80%～90% confluency, cells were passaged. Cells were incubated in the trypsinization mixture until all cells had rounded up, then resuspended in Keratinocyte-SFM and replated at a split ratio of 1/2.Origin assessment:The second passage human epithelial cells were seeded in 6-well plate with a coverslip in every well, cultured with K-SFM. These coverslips were fixed with alcohol of 95 percent for 10 minutes. Monoclonal mouse anti human Cytokeratin ( AE1/AE3 ) (Zhongshan, China) were used. Immunohistochemistry staining proceeded according to kit instructions. Blank control, negative control (fibroblasts) were set up at the same time. Observed under the light microscope, it could be judged as epithelial cells if brown gains found in its endochylema.Oral epithelial cells proliferation measurementXianhuayin decoction preparation:The effective component of nine kinds of Chinese medicinal materials mixture was precipitated with water and 95% alcohol. Then the 1g/ml Xianhuayin decoction was made by the means of aqueous bath. It was then decolored and filtered. Finally the Xianhuayin decoction was diluted with K-SFM to final concentrations of 10, 100, 500 and 1000μg/ml which were divided and stored at 4℃.The second passage oral epithelial cells were incubated in a 96-well plate. The wells were randomly divided into a control group and four experiments groups, with five wells in each group. After 72h the prepared Xianhuayin decoctions with concentrations of 0, 10, 100, 500 and 1000μg/ml were respectively added. The cells were cultured for another 5d. OD value was detected by means of the MTT colorimetric assay at 492 nm.2 Eighty cases of formal in-fixed, Paraffin-embedded specimens from oral mucosa lesions were obtained from the pathology archive files of the Department of Pathology of Hebei Medical University from 2002 to 2007. These specimens included 50 cases of OLP and 20 of OSCC. 10 cases of specimens from oral normal mucosa were taken as control. Immunohistochemical detection for PCNA was carried out for all specimens. The positive cells of PCNA were detected for oral normal mucosa, OLP and OSCC and analyze the contrast among them. Meanwhile, we compared the PCNA expression contrast between oral lichen planus with epithelial proliferation and oral lichen planus no epithelial proliferation. We compared the PCNA expression contrast between erosive oral lichen planus and non - erosive oral lichen planus .All statistical analyses were carried out with SPSS 13.0 and P<0.05 were considered significant.Result:1 Oral epithelial cell cultivationPrimary cultures: Observe on inverted phase contrast microscope, the isolated cells were variform. There were irregularly-shaped cells, large round cells and small round cells. The latter were healthy cells .Twenty four hours after seeding, a few cultured cells attached to the base of the culture flask. The cells grew and adhered continuely. Extended cells were predominantly small, polygonal and uniform with big nuclei that usually contained two nucleoli. In the third day, the quantity of cell increased obviously. After 7-day culture, the cell grew faster. Caryocinesis could be seen more than before. Cell entered exponential growth phase. Primary cultures were confluent after 2 week.When the cells reached 80%～90% confluency, the cells were passaged and reattached again after 24 hours. The second passage cells were similar to primary cultures, but grew faster than primary cells. Observed on inverted microscope, during the whole cell growing period, no fibroblast could be found and there were all simple epithelial cells.HE dyeing: Observation under light microscope, cell were polygonal .Nucelus and endochylema was well-distributed, the former was hepatic and the latter was pink. The cell body extended well. Nucleoli was clear. The cell linked each other showing mosaicism.Immunohistochemistry: Cultured cells were positive to broad range cytokeratin antibody. It strongly marked the cytoplasm with a tawny color.Effects of Xianhuayin on the proliferation in cultured human epithelial cells: The OD value of Xianhuayin decoctions with concentrations of 0, 10, 100, 500 and 1000μg/ml were 0.035, 0.067,0.117,0.053,0.042 respectively. By inspected the number of the cells with Xianhuayin decoctions with concentration of 100μg/ml was larger than that of the control group. The difference was significance (P<0.05), but the difference between concentrations of 10, 500, 1000μg/ml and the control group was no significance.2 The expression of PCNAIn human normal oral mucosa, PCNA positive cells stayed on basal layer and showed line arrange .The quantity of them is few. The other layer could be found positive reaction hardly. In oral lichen planus, PCNA expressed in the nuclear mainly. Them were distributed over basal layer mainly and sometimes could be found on underlayer of heckle cell .When oral lichen planus with epithelial proliferation, the PCNA positive cells could be found both on basal layer and underlayer of heckle cell. Compared with normal oral mucosa, the quantity of PCNA positive cells of OLP increased and color deepened. Compared with oral well-differentiated squamous cell carcinoma, the quantity of PCNA positive cells of OLP decreased, color shallowed and distribution range was smaller than oral well-differentiated squamous cell carcinoma obviously. The rates of positive nuclear PCNA expression in normal oral mucosa, OLP and oral well-differentiated squamous cell carcinoma tissue was different .The difference was statistically significant (P<0.05).The PCNA positive cells of non-erosive oral lichen planus centered in basal layer and vicinal layer of heckle cell. Compared with non-erosive oral lichen planus, the PCNA positive cells of erosive oral lichen planus positioned close surface layer. The rates of strong positive nuclear PCNA expression in them were 24% and 67%, respectively. The difference was statistically significant (P<0.05).The rates of positive nuclear PCNA expression in oral lichen planus with epithelial proliferation and oral lichen planus no epithelial proliferation and were 66%, 20%, respectively. The former was higher than the latter in proportion of positive PCNA expression. But both of them were lower than oral well-differentiated squamous cell carcinoma in shade and range of PCNA positive cells. The difference among them was statistically significant (P<0.05).Conclusion:1 Human oral epithelial cells have been successfully grown in serial culture with serum-free medium. 2 Xianhuayin had evident effect on promoting the growth of human oral epithelia cells on concentration of 100μg/ml in vitro.3 The rate of strong positive nuclear PCNA expression in OLP was higher than normal oral mucosa and lower than oral well-differentiated squamous cell carcinoma. It was indicated that OLP has the potency of canceration.4 The rate of strong positive nuclear PCNA expression in erosive oral lichen planus was higher than non-erosive oral lichen planus. It was indicated that erosive oral lichen planus should be taken care.5 The rate of positive nuclear PCNA expression in oral lichen planus with epithelial proliferation was higher than oral lichen planus no epithelial proliferation, but was lower than oral well-differentiated squamous cell carcinoma. Following the aggravation in the degree of histological anatomy, the rate of positive nuclear PCNA expression and the potency of malignancy increased.