Effects Of Carbon Dioxide Pneumoperitoneum On The Growth Of Human Cervical Carcinoma Cell Line In Vitro And Its Possible Mechanism

Despite the increasing prevalency of laparoscopic surgery for malignant tumors as an alternative to conventional surgical procedures due to less lesion, reduced post-operative pain, fewer postoperative complications, and shorter hospitalization , a burgeoning literature has suggested that laparoscopic manipulation of intraabdominal malignancies can lead to port site metastases in recent years. Although it is likely that the etiology of this phenomenon is of multiple factors such as tissue trauma at trocar sites and leakage of CO2 along trocar sites, aerosolization of tumor cells, the choice of the type of gases, and the influence of immune function, there is still a great concern that carbon dioxide (CO2), commonly used in laparoscopic procedures, may stimulate the growth and dissemination of tumor cells. Up to now , the impact of CO2 on the behavior of various tumor cells has been a matter of debate and the results of numerous experimental studies about the influence of CO2 insufflation on tumor growth remain discrepancy. Some authors showed an increase in cell proliferation and tumor growth, others found beneficial effects of CO2 exposure in vitro and in animal studies. To our knowledge, no experiment had been carried out to detect if the carbon dioxide pneumoperitoneum could stimulate human cervical carcinoma cell growth in vitro to date. In this experiment we established a laparoscopic pneumoperitoneum model in vitro and investigated the effects of carbon dioxide pneumoperitoneum with different pressures and different durations on the growths of human cervical carcinoma cell line(CaSki) in vitro and tried to elucidate the possible mechanism for providing initial assessment about the safety of CO2 pneumoperitoneum applied on gynecologic carcinoma.PART ONEESTABLISHMENT AND ASSESSMENT OF A LAPAROSCOPIC PNEUMOPERITONEUM MODLE IN VITROObjective:To establish and assess a laparoscopic pneumoperitoneum model in vitro.Methods:The model was established by a special airtight container made of stainless steel and an automatic pneumoperitoneum meter. After the cervical carcinoma cell line (CaSki) was exposed to 100% carbon dioxide in this model, the effect of carbon dioxide pneumoperitoneum on the cell growth was determined with MTT assay. And the experiment was repeated for three times.Results:There was no significant difference of the absorbance values at 24h,48h after exposure between the two groups(P>0.05),but at 72h, 96h,120h after exposure, the absorbance values in carbon dioxide pneumoperitoneum group were always significantly higher than those in control group at the same time(P<0.05). There was no significant difference of the change of the absorbance values either in the carbon dioxide pneumoperitoneum group or in the control group among three times(P>0.05).Conclusions:The model established with a special airtight container and an automatic pneumoperitoneum meter is an ideal one for research about laparoscopy in vitro,and the carbon dioxide pneumoperitoneum in this model increases the in vitro growth of human cervical carcinoma cells.PART TWO INFLUENCE OF CO2 PNEUMOPERITONEUM ON THE GROWTH OF HUMAN CERVICAL CARCINOMA CELLS IN VITROObjective:To evaluate the influence of CO2 pneumoperitoneum with different pressures and different durations on the growth of human cervical carcinoma cells in vitro.Methods:The CO2 pneumoperitoneum model was established in vitro. After CaSki cells were exposed to carbon dioxide pneumoperitoneum at 0mmHg,7mmHg or 14mmHg for 1h,2h,3h or 4h with the untreated group as control, the cell growth was determined by MTT assay , flow cytometry and electron microscopy.Results:1. The analysis of data by factorial experiment ANOVA show that both pressure and duration of CO2 pneumoperitoneum were the factors influencing the growth of CaSki cells, and the two factors were interacted with each other.2. The different cell growths curves of the groups dectected by MTT:2.1 The different cell growths curves of the groups at different CO2 duration under the same CO2 pressure: 1) Under 0mmHg, only the group with long duration (4h) could have a transient stimulating effect on the growth of CaSki cells. Under 7mmHg, the groups lasting 2~4h could stimulated the cell growth. However , Under 14mmHg, the groups with long duration (3~4h) could have stimulating effect on the growth of CaSki cells.2.2 The different cell growths curves of the groups under different CO2 pressure at same CO2 duration:When lasting 1h, the growth of CaSki cells was not influenced by the pressure of CO2 pneumoperitoneum. When lasting 2hs, only from 4th d to 5th d after treatment , the 7mmHg group show stimulating effect on the growth of CaSki cells. When lasting 3hs, both 7mmHg group and 14mmHg show stimulating effect, but the effect of 7mmHg group was stronger than that of 14mmHg group. When lasting 4hs, the growth of CaSki cells could be stimulated in all groups with different pressure , among which 7mmHg group was the strongest.3. The influence of CO2 pneumoperitoneum on the cell cycle of CaSki cells: When lasting 1~2h, the proportions of CaSki cells in G2-M phases under 7mmHg and 14mmHg were higer than those under 0mmHg or those in control group. And there was no significant difference between 7mmHg group and 14mmHg group. When lasting 3~4hs, the group under 14mmHg primarily increased the proportion of CaSki cells in S phase , but the group under 7mmHg primarily increased the proportion of CaSki cells in G2-M phases.4. The influences of CO2 pneumoperitoneum on the proliferation index (PI) of CaSki cells: When lasting 1~2h, there were no significant difference of PI among all groups. When lasting 3~4hs, the PI increased with the pressure of CO2 pneumoperitoneum being raised.5. The influences of CO2 pneumoperitoneum on the microstrucrures of the CaSki cells detected by electron microscopy : Common carcinoma cell characters were observed in those groups under 0mmHg and control group. However, active karyokinesis was observed in the groups under 7mmHg and 14mmHg.Conclusions:Within certain pressure and exposure duration scope, carbon dioxide pneumoperitoneum could increase the growth of human cervical carcinoma cells in vitro.PART THREE THE PROMOTING MECHANISM OF THE CO2 PNEUMOPERITONEUM ON THE GROWTH OF HUMAN CERVICAL CARCINOMA CELLSObjective: To study the effect of CO2 pneumoperitoneum with different pressures and durations on the expressions of proliferation-relative genes and apoptosis-relative genes in human cervical carcinoma cell line (CaSki) for elucidating its promoting mechanism on tumor growth.Methods: The CO2 pneumoperitoneum model was established in vitro. After CaSki cells were exposed to carbon dioxide pneumoperitoneum at 0mmHg,7mmHg or 14mmHg for 1h,2h,3h or 4h with the untreated group as control, the expression of Survivin and Caspase-3 mRNA were detected by RT-PCR; the expressions of Bcl-2 , Caspase-3 proteins were detected by Western blot; the expressions of Survivin, Bcl-2, Caspase-3, Ki-67 and PCNA proteins were detected by immunocytochemistry.Results:1. The analysis of data by factorial experiment ANOVA show that both pressure and duration of CO2 pneumoperitoneum were the factors influencing the expressions of Survivin, Bcl-2, Caspase-3, Ki-67 and PCNA in CaSki cells, and the two factors were interacted with each other.2. The results detected by immunocytochemistry show that Survivin,Bcl-2and Capase-3 mainly expressed at cytoplasm while PCNA and Ki-67 mainly expressed at cell nucleus.3. There was no significant difference of the expressions of Survivin among control group and 0mmHg ,14mmHg groups when CO2 pneumoperitoneum last 1~2h(P>0.05). The expressions of Survivin in other groups were significantly higher than those in control group(P<0.05). Under the same pressure, the expressions of Survivin in 3~4h groups were significantly higher than those in 1~2h group(sP<0.05). At the same exposure time, the expressions of Survivin in 7mmHg groups were always significantly higher than those in 0mmHg,14mmHg groups and control group(P<0.05).However, there was no significant difference of the expressions of Survivin between 0mmHg and 14mmHg groups(P>0.05).4. There was no significant difference of the expressions of Bcl-2 among control group and 0mmHg ,14mmHg groups when CO2 pneumoperitoneum last 1~2h(P>0.05). The expressions of other groups were significantly higher than those in control group(P<0.05). Under 7mmHg or 14mmHg, the expressions of Bcl-2 in 3~4h groups were significantly higher than those in 1~2h groups(P<0.05). At the same exposure time, the expressions of Bcl-2 in 7mmHg groups were always significantly higher than those in 0mmHg,14mmHg groups and control group(P<0.05).When lasting 1~2hs, there was no significant difference of the expressions of Bcl-2 between 0mmHg and 14mmHg groups(P>0.05).However, when lasting 3~4hs,the expressions of Bcl-2 in 14mmHg groups were significantly higher than those in 0mmHg groups(P<0.05).5. There was no significant difference of the expressions of Caspase-3 between 0mmHg groups and control group(P>0.05).However, the expressions of Caspase-3 in 7mmHg and 14mmHg groups were significantly lower than those in 0mmHg groups and control group(P<0.05). At the same exposure time, there was no significant difference of the expressions of Caspase-3 between 7mmHg and 14mmHg group(sP>0.05). Undere 0mmHg, there was no change of the expressions of Caspase-3 when exposure time prolonged(P>0.05).Under 7mmHg or 14mmHg, there was no significant differece of the expressions of Caspase-3 among 1~3h groups, but the expressions of Caspase-3 of 4h groups were significantly lower than those of 1~3h goups(P<0.05).6. There was no significant difference of the expressions of PCNA between 0mmHg groups and control group(P>0.05). The expressions of PCNA in 7mmHg and 14mmHg groups were significantly higher than those in 0mmHg groups and control groups, among which the expressions of PCNA in 14mmHg groups were the highest(P<0.05). There was no change of the expressions of PCNA in 0mmHg and 7mmHg groups when exposure time prolonged. Under 14mmHg,the expressions of PCNA of 3~4h groups were significantly higher than those of 1~2h groups(P<0.05).7. The expressions of Ki-67 in all of CO2 pneumoperitoneum groups were higher than those in control group(P<0.05).Under 0mmHg,there was no change of the expressions of Ki-67 when exposure time prolonged(P>0.05). Under 7mmHg or 14mmHg, the expressions of Ki-67 of 3~4h groups were significantly higher than those of 1~2h groups(P<0.05). The expressions of Ki-67 in 14mmHg groups were always higher than those in 0mmHg groups but lower than those in 7mmHg groups, among which, the expressions of Ki-67 in 7mmHg groups were the highest(P<0.05).Conclusions:1. Within certain pressure and exposure time scope, carbon dioxide pneumoperitoneums significantly up-regulated the expressions of Survivin, Bcl-2, Ki-67 and PCNA, but down-regulated the expression of Caspase-3 in CaSki cells.2. The promoting effect of carbon dioxide pneumoperitoneum on the growth of malignant cells is probably via increasing the capability of proliferation and anti-apoptosis of tumor cells.