| BackgroundLaparoscopy is a disruptive technology that has revolutionized surgery over the past few decades.Laparoscopic surgery has clear field of vision and is convenient for video recording,which is conducive to teaching and academic exchange.Cervical cancer is one of the most common malignant tumors in gynecology.Radical hysterectomy（RH）plus pelvic lymphadenectomy is the standard treatment for patients with early cervical cancer.A large number of studies have shown that laparoscopic cervical cancer surgery not only has less bleeding,short hospital stays,and low incidence of postoperative complications,but also a large number of studies have shown that laparoscopic cervical cancer surgery has similar oncology outcomes compared with open surgery.Laparoscopic cervical cancer surgery is therefore widely accepted and promoted,and was recommended by the National Comprehensive Cancer Network（NCCN）and International Federation of Obstetrics and Gynecology（FIGO）as alternatives to open cervical cancer surgery.However,the safety of laparoscopic surgery for cervical cancer remains controversial.In 2018,two studies published in the New England Journal of Medicine showed that laparoscopic cervical surgery had a higher mortality and recurrence rate than traditional open surgery.Based on this evidence,the 2021 NCCN guidelines indicate that an open approach is the standard procedure for radical hysterectomy of cervical cancer.This result overturns people’s previous understanding of laparoscopic surgery for cervical cancer,arouses great response in the field of gynecological oncology,and arouses heated discussion in the academic circles at home and abroad.There are a large number of cervical cancer patients in China,and the annual new cases account for 19%of the global total.It has become an urgent problem to explore the reasons for the high recurrence rate of cervical cancer by laparoscopic surgery and find solutions.Therefore,domestic and foreign experts held an in-depth discussion at the "International Conference on Clinical Research of laparoscopic Cervical Cancer Treatment" in October 2019,and believed that the possible reasons for the poor oncology outcome of laparoscopic cervical cancer surgery are as follows:The implementation of the principle of no tumor during surgery,the use of uterine manipulator,ultrasonic scalpel gasification,Carbon Dioxide（CO2）pneumoperitoneum,the way and method of vaginal dissection,and the experience and technical level of surgeons.This study will start from the use of ultrasonic scalpel and CO2 pneumoperitoneum,and attempt to explore the causes of poor oncology outcomes in patients undergoing laparoscopic cervical cancer surgery from clinical and basic perspectives,so as to provide decision-making basis for the selection of cervical cancer treatment.Chapter 1:Effect of laparoscopic ultrasonic scalpel on prognosis of patients with early-stage cervical cancerBackground and ObjectiveLaparoscopic surgery is highly dependent on instruments,and the application of various energy instruments promotes the development of minimally invasive gynecological surgery.Ultrasound scalpel has both cutting and coagulation functions and little lateral thermal damage,so it has gradually become the first choice for most laparoscopic cervical cancer surgery.The ultrasonic scalpel cuts the tissue by highfrequency vibration,and the tissue fragments will fly away in the form of air mist under the action of the shock of the knife head.The temperature of ultrasonic scalpel cutting is only 40℃～100℃.Studies have shown that the aerosol generated when the ultrasonic knife is stimulated can carry active tumor cells.So,is there a correlation between the use of ultrasound scalpel in laparoscopic radical hysterectomy and poor oncology outcome of laparoscopic cervical cancer surgery?This study compared the clinical efficacy of RH using ultrasonic scalpel in laparoscopic surgery with EnSeal and traditional open surgery,to explore the impact of laparoscopic ultrasonic scalpel on the prognosis of patients with early cervical cancer.MethodsClinical data of patients who underwent RH in Qianfoshan Hospital from January 2013 to December 2018 were retrospectively analyzed.Patients were divided into three groups according to different surgical approaches and surgical instruments including:LRH EnSeal（LRH-E）group,LRH ultrasonic scalpel（LRH-U）group and ARH group.Progression-free survival（PFS）,overall survival（OS）,duration of surgery,estimated blood loss,transfusion rate,postoperative exhaust time,number of retroperitoneal lymph nodes resected,and incidence of postoperative Lymphocele were compared.ResultsA total of 117 patients were included,including 35 patients in the LRH-E group,46 patients in the LRH-U group and 36 patients in the ARH group.There were no significant differences in age,weight,FIGO stage,pathological type,tumor grade,tumor size and adjuvant therapy rate among the three groups（P>0.05）.The LRH-E group,LRH-U group and ARH group were followed up for 9～71 months,5～87 months and 15～95 months.Kaplan-Meier survival analysis showed that there was no significant difference in PFS and OS among the three groups（P>0.05）.The operation time and estimated blood loss in LRH-E group were less than those in LRH-U and ARH groups.There was no significant difference between the LRH-E group and the LRH-U group in postoperative exhaust time and number of lymph nodes removed（P>0.05）.The postoperative exhaust time in the LRH-E and LRH-U groups was less than that in the ARH group（P<0.05）,and the number of lymph nodes was more than that in the ARH group（P<0.05）.In addition,there was no significant difference in the incidence of lymphocele among the three groups（P>0.05）.ConclusionIn patients with early-stage cervical cancer,LRH with ultrasound scalpel does not increase the risk of tumor metastasis and recurrence.Chapter 2:Effects of CO2 pneumoperitoneum on proliferation and apoptosis of cervical cancer cells and xenograft tumor in nude miceBackground and ObjectiveThe difference between laparoscopy and traditional open surgery is the need to establish artificial pneumoperitoneum.CO2 is the preferred medium for laparoscopic pneumoperitoneum.It is not only cheap,easy to obtain,and non-flammable,but also highly soluble in the blood and can be quickly absorbed and excreted from the body.Studies have shown that CO2 pneumoperitoneum is closely related to malignant tumor metastasis and puncture site implantation,but the effect of CO2 pneumoperitoneum on tumor cell proliferation is controversial,and there are few studies on the effect of CO2 pneumoperitoneum on cervical cancer cells.This part mainly studies the effect of CO2 pneumoperitoneum on the proliferation and apoptosis of cervical cancer cells and tumor-formation in nude mice,and discusses the effect of CO2 pneumoperitoneum on the prognosis of cervical cancer patients undergoing laparoscopic surgery.MethodsThe CO2 pneumoperitoneum model was established by using polypropylene sealed box and Olympus pneumoperitoneum machine.Cervical cancer cell lines HeLa and SiHa were selected and divided into CO2 group and control group.Cells in the CO2 group were stimulated with CO2 pneumoperitoneum model at 13mmHg for 4 h,and cells in the control group were cultured under conventional conditions.The proliferation and tumorigenesis of cervical cancer cells were detected by CCK-8 method,clone formation assay and subcutaneous tumorigenesis assay in nude mice.Cells were stained with FITC-Annexin V and PI,and cell apoptosis rate and cell cycle distribution were determined by flow cytometry.A CO2 pneumoperitoneum model was established in mice.HeLa cells were injected intraperitoneally and treated with CO2 pneumoperitoneum（6mmHg）for 60 minutes.4 weeks later,the mice were sacrificed and dissected,and the abdominal metastatic tumor was stripped and weighed,and the Experimental Peritoneal Carcinomatosis Index（ePCI）and tumor load were observed and calculated.ResultsCCK-8 assay showed that the cell viability of HeLa and SiHa cells in CO2 group decreased at the initial stage,the cell proliferation activity increased significantly at the later stage.From day 4,the proliferation activity of HeLa cells was significantly higher than that of control group（P<0.05）,the proliferation activity of SiHa cells from day 5 was significantly higher than that of control group（P<0.05）.The results of clone formation experiment showed that after CO2 pneumoperitoneum stimulation,the plate clone formation rate of HeLa and SiHa cells increased from 19.9%and 22.5%to 28.7%and 29.7%,respectively（P<0.05）.24 hours after CO2 stimulation,the apoptosis rates of HeLa（SiHa）cells in CO2 group and control group were 12.4%（10.6%）and 6.2%（8.0%）,respectively.HeLa cells in G2 phase of CO2 group increased from 14.0%±0.7%to 19.2%±0.4%（P<0.05）.The number of SiHa cells in G2 phase increased from 15.6%± 0.7%to 19.4%± 0.5%（P<0.01）.After stimulation in nude mice with CO2 pneumoperitoneum model（6mmHg,60 min）,the tumor load in CO2 group was significantly higher than that in control group（P<0.01）,ePCI score in CO2 group was significantly higher than that in control group（P<0.05）.Conclusion1.CO2 pneumoperitoneum in vitro promoted the proliferation of cervical cancer cell lines HeLa and SiHa,and promoted the growth of subcutaneous transplanted tumor.2.In vivo CO2 pneumoperitoneum can promote the growth of cervical cancer HeLa cells in the abdominal cavity of nude mice and promote tumor spread.Chapter 3:A preliminary study on proteomics and molecular mechanism of CO2 pneumoperitoneum on proliferation of cervical cancer cellsBackground and ObjectiveCO2 pneumoperitoneum will not only have adverse effects on patients’ respiratory,circulatory,urinary systems,but also have a wide impact on the growth and metastasis of tumor cells.The results of the second part of this study show that CO2 pneumoperitoneum can promote the proliferation of cervical cancer cells and the formation of subcutaneous and abdominal tumorigenesis in mice,and the molecular mechanism is still unclear.CO2 pneumoperitoneum can cause hypoxia and acidification of cell environment simultaneously,and the effect on biological function of cervical cancer cells is multi-factors.Proteomics can analyze samples with high throughput and high efficiency.In this part,the effect of CO2 pneumoperitoneum on cervical cancer cells was studied from the protein level by using TMT based proteomics technology,and the possible regulatory pathway of CO2 pneumoperitoneum promoting cervical cancer cell proliferation was explored.MethodsHeLa cells were collected after stimulated with an in vitro simulated CO2 pneumoperitoneum model（13mmHg,4 h）.Cell proteins were extracted and digested into peptides by FASP method.They were labeled with a TMT mass tagging kits and reagents,graded with a high pH reverse-phase peptide fractionation kit,and analyzed by mass spectrometry with Q-Exactive mass spectrometer.Mascot 2.2 and Proteome Discoverer 1.4 were used for library identification and quantitative analysis.Differentially expressed proteins were obtained with expression multiple>1.2 times（up-regulated>1.2 times or down-regulated<0.83 times）and P<0.05（T-test or other criteria）.The Complexheatmap R package was used for cluster analysis,the method of CELLO was used for subcellular localization prediction.InterProScan software package and Pfam database were used for protein domain analysis.Blast2GO was used for GO annotation of target protein set.KAAS software was used to annotate the KEGG pathway of the target protein set.The target protein set was enriched by GO annotation or KEGG annotation using Fisher’s exact test.pH values of extracellular fluid were measured before and after CO2 pneumoperitoneum stimulation in vitro.In this study,we simulated hypoxic and acidic conditions caused by CO2,and used CCK-8 method to study the different effects of the two factors on the proliferation of cervical cancer cells.Western blot was used to detect the changes of cell proliferation and PI3K/Akt pathway-related protein expression after CO2 stimulation and addition of PI3K pathway inhibitors.The effect of PI3K inhibitor LY294002 on tumor formation in abdominal cavity was studied by constructing CO2 pneumoperitoneum model in nude mice.ResultsIn vitro CO2 pneumoperitoneum induced changes in protein expression of HeLa cells,which affected the biological function of cervical cancer cells.A total of 160273 spectrograms,64,387 identified peptides and 6778 identified proteins were obtained in this project.A total of 177 differentially expressed proteins were identified,including 115 up-regulated proteins and 62 down-regulated proteins.Subcellular localization analysis showed that 11.9%of differentially expressed proteins were related to mitochondria,and domain analysis showed that CHCH structures were significantly enriched.The results of GO functional analysis and enrichment analysis suggest that positive regulation of cell adhesion mediated by integrin,cell adhesion mediated by integrin,regulation of cell adhesion mediated by integrin,myeloid leukocyte activation,extracellular matrix organization and other important biological processes proteins were significantly changed.KEGG pathway analysis and enrichment analysis showed that the proteins in pathways of p53 signaling pathway,beta-Alanine metabolism,ECM-receptor interaction,PI3K-Akt signaling pathway and oxidative phosphorylation were significantly enriched.After CO2 pneumoperitoneum treatment,the pH value of extracellular fluid decreased rapidly.The higher the pneumoperitoneum pressure is,the faster pH decreases.After the pneumoperitoneum stopped,the pH value of extracellular fluid gradually increased and returned to the level of the control group 5 hours later.CCK-8 assay showed that extracellular fluid acidification inhibited cell proliferation and hypoxia promoted cell proliferation.The expression level of p-Akt and proliferation activity were significantly decreased after PI3K inhibitor was used（P<0.05）.Compared with the control group,the ePCI score of nude mice in CO2 group was significantly higher than that in the control group（P<0.05）,ePCI score of nude mice in CO2+LY294002 group had no significant change.After the nude mice were sacrificed,all tumors in the abdominal cavity were stripped and weighed,and the tumor load in the CO2 group was significantly higher than that in the control group（P<0.05）,while the tumor load in CO2+LY294002 group was significantly lower than that in CO2 group（P<0.05）Conclusion1.In vitro simulated CO2 pneumoperitoneum affects the biological behavior of cervical cancer cell line HeLa cells through cell hypoxia and cell acidification.2.CO2 pneumoperitoneum may promote cell proliferation of cervical cancer HeLa cell by activating PI3K/Akt signaling pathway. |
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