Expression Of Survivin, PTEN Genes In Osteosarcoma And Its Relation With Apoptosis, Proliferation

The occurrence of osteosarcoma is a multi-stage, multi-step process in which exists the disturbance of multi-gene regulation. On one hand, it is related to the overexpression of oncogene that causes cellular proliferation and inhibits cellular apoptosis. On the other hand, it is related to the variance of incogene that induces cell apoptosis. That is to say the occurrence of osteosarcoma is related to the cell abnormal proliferation and the disturbance of cellular apoptosis. Survivin protein, found recently in an inhibition of apoptosis protein (IAP) family, has a special construction. It can protect the particular cell resisting a series stimulus zymogen inducing, inhibit the apoptosis, and be closely related to the malignant degree of various tumors and prognosis. Survivin gene was regarded as one of new oncogenes. The development of the Survivin biology should be combined with the specificity caspase family member in various ways, inhibit its activity, and obstruct the signal conduction of dependence of caspase family member cell apoptosis.PTEN is the first tumor suppressor gene that can encode a dual specificity lipid and protein phosphatase enzyme. As a kind of multi-function protein, PTEN protein negatively regulates andcauses cell apoptosis through PI3K-Akt/PKB signal transmission pathways, and antagonizes PTKS to inhibit cell proliferation.Ki-67 protein is a kind of nuclear proteins related to the cell proliferation. It is indicated that it not only can be used as the norm evaluating cell propagated state, but also be the norm judging the tumor prognosis.Cell apoptosis plays an important role in the multi-cell organism growth, organic steady state, tumor surveillance and immunity system. It is one of the mechanisms, which maintain human normal cell. It is also indicated that the disturbance of cellular apoptosis is associated with many diseases, especially with the tumors.There is little research on the relationship between the biology behavior of the osteosarcoma of Survivin and PTEN, neither their relativity between the cell proliferation and apoptosis. In this study, we adopt TUNEL technique to identify cell apoptosis and its special distributing in the osteosarcoma and osteochondroma. And we adopt the immunohistochemistry streptomycin -avidin -peroxide-enzyme labeling to test the three gene protein expression in person's osteosarcoma, osteochondroma tissue, to analyze the relativity of Survivin, PTEN, ki-67 proteins expression and pathological characters, and to research the relativity of Survivin, PTEN proteins expression and apoptosis, cell proliferation.Material and method:(l) The tissue specimen were drawn from the clinical operation of return back to the file wax piece from Department of Pathology in Henan Provincial People's Hospital from 1996 to 2003. The selected 43 osteosarcomas and 15 osteochondromas specimens were soaked in 10% neutral formaldehyde solution and paraffin wax embedding after routine treatment. (2) Immunohistochemistry streptomycin -avidin -peroxide- enzyme labeling as taken to test expression circumstancesof Survivin, PTEN and ki-67 in osteosarcoma tissue, osteochondroma tissue. (3) TUNEL reagent box was taken to identify apoptosis cell. (4) SPSS 10.0 version software bag was used in statistics process. Group rates x2 test was used to compare two sample rates, spearman grade was used to analyze the correlation, a =0.05 check standard was used to check the divided data of experiment.Results: (1) In 15 osteochondroma specimens, AI (apoptosis index) was (16.18 + 3.26)%, but in 43 osteosarcoma specimens, AI was (5.87 + 2.07)%. AI was significantly different between the osteochondroma group and osteosarcoma group. AI of Enneking II group was (7.22+1.01)%, but EnnekingUI were (3.59+1.17)%. There was significant difference between two groups. According to histological type, AI in the type of osteoblast, chondroblast, the type of fibroblast and mixing type were (5.94 + 2.23)%, (6.20 + 2.03)%, (6.08 + 2.10)%, (4.97+1.82)% respectively. There was no significant difference among deferent groups by statistics analysis.(2)In 15 osteochondroma specimens, ki-67 index was (2.18 + 0.84)%, but in 43 osteosarcoma specimens, ki-67 index was (27.08 + 9.45)%. The ki-67 index was significantly different between the osteochondroma group and osteosarcoma group(P<0.01). ki-67 index of Enneking II group was (20.65 + 4.64)%, but EnnekingUI was (37.94 + 3.50)%. There was significant difference between two groups (P<0.01). ki-67 index in the type of osteoblast, chondroblast, the type of fibroblast and mixing type were (26.60+10.33)%, (28.24 + 9.40)%, (27.15 + 9.96)%, (26.90 + 8.31)% respectively. There was significant difference among deferent groups by statistics analysis.(3) In 43 osteosarcoma specimens investigated, Survivin protein positive expression rate was 69.44%(29/43). In 15 osteochondromaspecimens, there was no survivin protein positive expression. There was significant statistic difference in the two groups (P<0.001). Among Enneking II group and EnnekingUI group of osteosarcoma, survivin protein positive rate were 51.85%( 14/27) and 93.75%(15/16). There was significant difference between two groups. According to histological type, osteosarcoma was divided into the type of osteoblast, the type of chondroblast, the type of fibroblast and mixing type, etc. The positive expression rate of the above three types was 68.75%, 71.43%, 69.23%, and 57.14% respectively with no significant difference (P>0.10).(4) In 43 osteosarcoma specimens investigated, AI and KI were (4.89+1.76)% and (29.93+9.73)% respectively in the positive expression group of survivin protein, but in the negative expression group of survivin protein, AI and KI were (7.90 + 0.77)% and (21.18+11.70)% respectively. AI and KI were significantly different between the two groups by statistics analysis (P<0.05). According to Spearman grade correlation analysis examines, survivin protein expression was negatively related to AI, but it was positively related to KI.(5) The PTEN protein positive rate was correspondingly 76.74%(33/43) and 93.33%(14/15) in 43 osteosarcoma and 15 osteochondroma specimens. Comparing the staining positive rate of the two groups, there was no significant difference between the two groups(/>>0.05). PTEN protein positive rate of Enneking II group was 81.48%(22/27), III group was 68.75%(11/16), comparing Enneking II group and EnnekingUI group, stained positive rate showed descent trend, but there was no significant difference between the two groups by statistics analysis (P>0.05). Positive expression rate of protein PTEN in the type of osteoblast, chondroblast, the type of fibroblast and mixing type were 81.25%,71.43%, 76.92%, 71.43%. There was no significant difference among histological sub-type (P>0.10).(6) In 43 osteosarcoma specimens investigated, AI and KI were (5.79 + 2.17)% and (25.05 + 8.95)% in the positive expression group of PTEN protein respectively, but in the negative expression group of PTEN protein, AI and KI were (6.14+1.77)% and (33.77 + 8.18)% respectively. KI was significantly different between the two groups by statistics analysis (P<0.05). While AI was not significantly different between the two groups. According to Spearman grade correlation analysis examinations, PTEN protein expression was negatively related to KI, but it was not related to AI.(7) In osteosarcoma tissue of positive PTEN protein expression, positive expression rate of survivin gene was 57.58%(19/33). In osteosarcoma tissue of negative PTEN protein expression, positive expression rate of survivin gene was 100%( 10/10). Survivin gene of PTEN protein positive expression group was higher than that of PTEN protein negative expression group. Using spearman grade correlation analysis examination, there was obviously negative relativity between them.Conclusion(l) AI in the osteosarcoma was obviously lower than that in the osteochondroma, and AI of Enneking II group was higher than that of Enneking III group in osteosarcoma. It is indicates that cellular apoptosis turbulence exists in the progression of osteosarcoma occurrence.(2) Ki-67 is a protein that is related to cell proliferation. The expression of the ki-67 protein in osteosarcoma is closely related to the grade extent of pathology of tumor Enneking phrase, and it is an important cell proliferation norm. As a marking that reflects cell proliferation, testing the expression of the ki-67 can reflect the circumstance of tumor cell proliferation, and can be used as a normthat evaluates the circumstance of cell proliferation. It might be an important norm that judges the occurrence, development and prognosis of a tumor.(3) Expression of the survivin gene protein is closely related to the occurrence and development of osteosarcoma and Enneking phrase, but has no relation to the histology sub-type. So the survivin protein can be used as a good norm that judges the occurrence and development of osteosarcoma, and provides the theoretical foundation for gene therapy, which regards the survivin gene as the target.(4) In osteosarcoma tissues, apoptosis cells mainly concentrate in the group in which survivin proteins express negatively. AI in survivin protein positive group was obviously higher than that in negative group, KI was also significantly different between the two groups by statistics analysis. Survivin protein expression was negatively related to AI, but it was positively related to KI.(5) Loss of PTEN expression existed in osteosarcoma, which was not associated with osteosarcoma progression. PTEN protein expression was not related to biologic behavior character of osteosarcoma. It is only one of these factors which influence multi-stage osteosarcoma progression. And it probably took part in the occurrence, development of osteosarcoma.(6) PTEN protein expression was negatively related to KI, but it was not related to AI. These data indicate that PTEN protein doesn't inhibit tumor cell apoptosis through PTEN/Akt signal transmissional pathways in osteosarcoma.(7) In osteosarcoma specimens, with osteosarcoma differentiation deteriorate, survivin protein positive rate raises and PTEN protein positive rate reduces gradually. According to Spearman grade correlation analysis examination, surviving proteinexpression was negative related to PTEN protein.