Abstract Objective: To study the antitumor effect of CD3AK (CD3 activated killer cells) in pelvic drainage lymph nodes of ovarian cancer activated by CD3 monoclonal antibody in vitro. Methods: The lymphocytes of pelvic drainage lymph nodes of ovarian cancer patients were induced and activated in vitro with CD3 monoclonal antibody and a small amount of rIL-2. Experimental studies were conducted on two human ovarian cancer HO-8910 and HO-8910PM cell lines. Randomly divide the two cells into 6 groups: (1) negative control group (group A): only add fresh whole culture solution; (2) low concentration CD3AK group (group B): 5 × 105.L-1 CD3AK culture ; (3) Medium concentration CD3AK group (C group): 1 × 109.L-1 CD3AK culture solution; (4) High concentration CD3AK group (C group): 2.5 × 109.L-1 CD3AK culture solution; (5) Cisplatin positive control group (group E): containing 10 mg.L-1 cisplatin culture medium; (6) cisplatin + CD3AK combination medication group (group F): containing 10 mg.L-1cisplatin + 1 × 109 .L-1 CD3AK culture solution was observed by LDH activity measurement, natural killing rate and trypan blue live cell counting method. Results: CD3AK cells had an obvious killing effect on ovarian cancer cells. Both cells showed good cytotoxic activity at medium concentration of CD3AK cells (P <0.01), and the difference was highly statistically significant. When CD3AK cells were used in combination with cisplatin, no matter the cell proliferation inhibition LDH activity (OD value) measurement or natural killing rate showed a significant cytotoxic activity on both cells (P <0.001). Conclusion: CD3AK cells have a killing effect on human ovarian cancer cells, and the combined use of CD3AK and cisplatin has a good synergistic anti-ovarian cancer effect. Keywords ovarian tumor; lymph node; antibody, CD3; cell line China Book Classification Number R737.31; R322.25; R730.51 Document Identification Code A Article Number 1001-7399 (1999) 01-0054-03 The kill effect of CD3AK cells on human ovarian cancer cell line Qian Lijuan, Xu Shenhua, Mou Hanzhou et al (Zhejiang Cancer Research Institute, Hangzhou 310022) ABSTRACT Purpose In order to study anti-tumor effects of lymphocytes of draining lymph nodes in pelvic cavities in patients with ovarian cancer, which were activated by CD3McAb in vitro. Methods Both two cell lines were divided into 6 groups respectively and randomly: (1) Negative contrast group (A): only adding fresh complete culture medium; (2) Low consistency CD3AK group (B): 5 × 105.L-1 CD3AK culture medium; (3) Middle consistency CD3AK group (C): 1 × 109 .L-1 CD3AK culture medium; (4) High consistency CD3AK group (D): 2.5 × 109.L-1 CD3AK culture medium; (5) Cisplatin positive contrast group (E): 10 mg.L-1 cisplatin culture medium ; (6) Usage of combination of cisplatin + CD3AK group (F): 10 mg.L-1 cisplatin + 1 × 109.L-1 CD3AK culture medium. In this study, LDH level in the culture fluid and cellular count were used to investigate the kill effects. Results The results showed that CD3AK cells had obvious kill effects on ovarian cancer cells. Middle consistency CD3AK cells showed cell toxic effects on two cell lines. The usage of combination of cisplation + CD3AK group had stronger kill effects than that of cisplatin group alone (P <0.01). Conclusion CD3AK has kill effects on ovarian cancer cells. The usage of combination of cisplatin + CD3AK have cooperative resist effects on ovarian cancer. KEY WORDS ovarian neoplasms; lymph nodes; antibady, CD3; cell line CD3AK (CD3 activated killer cells) is a killer cell activated by anti-CD3 monoclonal antibody. Its proliferative ability and anti-tumor cytotoxic activity are significantly better than LAK cells, so it is increasingly valued [1 to 3]. In this experiment, the lymphocytes isolated from the pelvic drainage lymph nodes of human ovarian cancer patients were used to induce the activation of CD3 monoclonal antibodies. On the self-built human ovarian cancer cell line, the in vitro killing effect of CD3AK cells on human ovarian cancer cells was studied. 1 Materials and methods 1.1 The cell-derived human ovarian cancer HO-8910 cell line was established by the Institute in 1989 [4]: ​​The high-metastatic human ovarian cancer HO-8910PM cell line was also inoculated from HO-8910 cells in nude mice and subcultured to lung metastases in vitro Established after cultivation. Both cells were passaged in vitro as usual, and passaged or fluid changed twice a week. 1.2 The CD3 monoclonal antibody derived from antibodies and IL-2 is of murine origin and is a product of the Biological Preparation Development Center of the Academy of Military Medical Sciences; genetically recombinant IL-2 produced by Beijing Sihuan Bioengineering Products Factory (supervised by the Institute of Bioengineering, Academy of Military Medical Sciences). 1.3 Cell culture conditions: RPMI-1640 medium made in the United States is supplemented with 15% heat-inactivated fetal bovine serum (product of Hangzhou Sijiqing Bioengineering Materials Research Institute). Each ml contains penicillin 100 U, streptomycin 100 μg, insulin 0.01 U, Hydrocortisone 10 μg. Incubate at 37 ° C and 5% CO2. 1.4 Induction of CD3AK Several pelvic dissection lymph nodes of patients with ovarian cancer were taken under sterile conditions during operation, and half of each was sent for pathological examination. The half of the lymph nodes were mechanically shredded on a 280 mesh Tan mesh and gently ground, and tea was used Filter paper to make a single cell suspension, and then use lymphocyte separation liquid to separate lymphocytes, adjust the cells to 1 × 109.L-1 in the culture medium containing 0.5 mg.L-1 CD3 monoclonal antibody, and set at 37 ℃ 5 Cultivate in a CO2 incubator for 24 h, centrifuge to remove the supernatant, re-add fresh culture medium containing 0.5 mg.L-1 CD3 monoclonal antibody and 5 × 104 UL-1 recombinant human IL-2, and continue culturing for 5 days. It is used for the experiment of killing and inhibiting human ovarian cancer cells in vitro. 1.5 Experimental group Take two well-grown HO-8910 and HO-8910PM cells, digest them to make 1 × 108.L-1 3 ml bottles, incubate at 37 ℃, 5% CO2 incubator for 72 h, and randomly divide them into 6 groups: (1) negative control group (group A) only add fresh whole culture medium; (2) low concentration CD3AK group (group B): 1 × 108.L-1 CD3AK culture medium; (3) medium concentration CD3AK group (Group C): 1 × 109.L-1 CD3AK medium; (4) High concentration CD3AK group (Group D): 2.5 × 109.L-1 CD3AK medium; (5) Cisplatin positive control (Group E) : Contains 10 mg.L-1 cisplatin culture solution; (6) Cisplatin + CD3AK combination medication group (F group): Contains 10 mg.L-1 cisplatin + 1 × 109.L-1 CD3AK culture solution, wait After 72 hours of culture, the original culture solution was discarded, and different culture solutions were changed into groups. The negative control group was also changed, and the results were observed after 96 hours of incubation. 1.6 Detection of LDH in the experimental cell supernatant Two groups of cells were taken and the supernatant was taken. According to the method of literature [5], the wavelength was 492 nm using a microplate reader (Clini Biol28, product of SLT Labinstrument, USA), and 5 in each group Double holes, calculate the average value, and calculate the natural killing rate according to the formula. Natural kill rate (%) = 5501.gif (1914 bytes) 1.7 Trypan blue exclusion method live cell counting Two cells of each group were counted, and the average value was obtained by repeating 4 times. 2 results 2.1 The effect of CD3AK on LDH of ovarian cancer cells From Table 1, it can be seen that CD3AK cells have a certain cytotoxic activity on two ovarian cancer cells. The three dilution concentrations set (1: 5, 1:10, 1:25 ) At moderate concentration, it showed good cytotoxic activity, HO-8910 cells C and D compared with group A were P <0.01 and P <0.05, HO-8910PM cells C and D compared with group A were: P <0.01 and P <0.05. Compared with group A, both cells of group F had P <0.01, while group F compared with group E, HO-8910 cells had no significant difference, and HO-8910PM cells had P <0.01. From the point of view of natural killing rate of cells, it increased with the increase of CD3AK concentration, however, the combination group showed a higher killing rate in both cells, which was stronger than the positive control group using cisplatin alone CD3AK has a good synergistic effect of cisplatin against ovarian cancer. Table 1 Effect of CD3AK on LDH of HO-8910 and HO-8910PM cell lines Group HO-8910 cells HO-8910PM cells OD value (1.gif (65 bytes) ± s) Natural kill rate (%) P value OD value (1.gif (65 bytes) ± s) natural kill rate (%) P value Negative control group (A) 0.100 ± 0.0350 0.100 ± 0.0349 CD3AK low concentration group (B) 0.128 ± 0.0042 23 <0.01 0.122 ± 0.0691 18> 0.05 CD3AK medium concentration group (C) 0.129 ± 0.0025 24 <0.01 0.120 ± 0.0237 17 <0.05 CD3AK high concentration group (D) 0.134 ± 0.0168 28 <0.01 0.130 ± 0.0255 25 <0.05 Cisplatin positive control group (E) 0.190 ± 0.0961 75 <0.01 0.190 ± 0.0150 75 <0.01 Combined medication group (F) 0.200 ± 0.0559 83 <0.001 0.209 ± 0.0199 90 <0.001 ★ Comparison between Group F and Group E: HO-8910 P> 0.05, HO-8910PM P <0.01 2.2 The effect of CD3AK on the inhibition of ovarian cancer cell proliferation using trypan blue exclusion method is shown in Table 2. The inhibitory effect of CD3AK on ovarian cancer cells increases with the increase of CD3AK concentration. Compared with group A, P <0.05 and P <0.01, HO-8910PM cells C and D compared with group A had P <0.01. The combination of the two cells in the combination treatment group had the most obvious cell suppression effect, both of which were fewer than the cisplatin positive control group, but there was no statistically significant difference between the two. Table 2 The effect of CD3AK on the proliferation inhibition of HO-8910 and HO-8910PM cells Group HO-8910 cells HO-8910PM cells Cell number (1 × 105) (1.gif (65 bytes) ± s) P value cell number (1 × 105) (1.gif (65 bytes) ± s) P value Negative control group (A) 85 ± 12.12 113.5 ± 17.7 CD3AK low concentration group (B) 76.5 ± 14.38> 0.05 108.5 ± 16.12> 0.05 CD3AK medium concentration group (C) 71.5 ± 10.9 <0.05 90 ± 12.65 <0.01 CD3AK high concentration group (D) 71.8 ± 7 <0.01 79.5 ± 11.94 <0.001 Cisplatin positive control group (E) 10 ± 6.32 <0.001 6.5 ± 4.16 <0.001 Combined medication group (F) ★ 5.5 ± 3.82 <0.001 4 ± 4.32 <0.001 ★ Comparison between group F and group E: both cells P> 0.05 3 Discussion CD3 is a differentiation antigen on T lymphocytes. It forms a TCR-CD3 complex with T cell antigen receptors and plays an important role in the recognition and activation of T cells [6]. Experiments show that almost all peripheral blood T cells can be activated by CD3 antibodies, and the activated cells are significantly higher than LAK cells. A large number of experimental studies and LAK cells show that CD3AK cells have a stronger anti-tumor effect in vivo and in vivo [1, 7 ,8〕. We used lymphocytes from the pelvic drainage lymph nodes of human ovarian cancer patients, in vitro activation after in vitro activation with CD3 monoclonal antibody in nude mice carrying high-metastatic human ovarian cancer, and showed that CD3AK cells have anti-tumor growth and anti-metastatic effects In the CD3AK treatment group, 1/7 nude mice had tumor regression, and only 2/7 nude mice had metastasis, which was significantly different from that of 8/10 nude mice in the control group (P <0.05) [9]. On the basis of this work, the killer cells CD3AK induced and activated by the same method were used in three different dilution concentrations to conduct preliminary experimental studies on two of our self-built human ovarian cancer cell lines HO-8910 and HO-8910PM , And using cisplatin as a positive control, the results show that: CD3AK cells have a certain killing effect on ovarian cancer cells in vitro, CD3AK cells with a medium concentration showed a good cytotoxic activity, statistically significant differences. The combination drug group showed significant cytotoxic activity on two ovarian cancer cells regardless of cell proliferation inhibition, LDH activity (OD value) measurement or natural killing rate, and this cytotoxic activity was found in HO-8910 and HO There is no obvious difference between -8910PM cells. The experimental results suggest that CD3AK can inhibit the growth of ovarian cancer cells, and it has a more significant synergistic anti-ovarian cancer effect in combination with cisplatin. In summary, CD3AK cells induced and activated by ovarian cancer pelvic drainage lymph nodes induced by CD3 and IL-2 can effectively inhibit the growth of ovarian cancer cells and show cytotoxic activity. This cytotoxic activity may be dependent on PKC related. PKC is an important substance in the transmission of T cell activation information. CD3AK cells activate T cells through PKC to activate a series of substrate enzymes, thereby starting T cell activation and proliferation and exerting anti-tumor activity. In-depth research will provide a basis for the use of CD3AK cells for anti-tumor immunotherapy in patients with clinical ovarian cancer or other tumors. Mini Dune Buggy,Small Dune Buggy,Mini Rail Buggy,Mini Rail Buggy Binzhou Daowang Power Co.,Ltd , https://www.dwutv.com