Combined extract of heated TC1, a heat-killed preparation of Lactobacillus casei and alpha-galactosyl ceramide in a mouse model of cervical cancer

Reagents

The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Griess Reagent Kit, Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Dimethyl sulfoxide (DMSO), and Phosphate-buffered saline (PBS) were acquired from Kalazist (Tehran, Iran). From Karmania Pars Gene (Kerman, Iran), ELISA kits for enzyme-linked immunosorbent assays (ELISA) were ordered.

Cell culture

We received TC1 cells from the Baqiyatallah University of Medical Sciences of Iran. The cells were maintained in monolayer cultures in DMEM supplemented with 10% FBS and cultivated at 37 °C in a humidified atmosphere with 5% CO2 (Fig. 1) [12].

Fig. 1figure 1

TC1 cell line growth under cell culture condition. A 3 days after cell culture, B 7 days after cell culture

Bacterial strains and growth conditions

L. casei (ATCC:393) was purchased from the central laboratory of Baqiyatallah University of Medical Sciences of Iran. A Rogosa’s medium was applied to cultivate the bacteria. The process was performed at 37 °C for 24 h [13].

Preparation of cell and bacterial extracts

In order to obtain cell extracts, 104 TC1 cells were exposed to non-lethal excessive heat (43 °C, 30 min), and freeze-and-thaw (− 196 °C, 30 min). The cells were then centrifuged and utilized as a killed cell extract in the treatment of cancerous mice. Additionally, 3 × 108 CFU/ml of grown bacteria were heated at 56 °C for 60 min in order to obtain the bacterial extract, which was subsequently centrifuged for use in the current investigation [13].

Experimental design, mice and tumor induction

From Iran's Baqiyatallah University of Medical Sciences, 80 C57BL/6 female mice aged 6 to 8 weeks were purchased. Standard housing for the mice included a temperature range of 22–24 °C, 12-h light/dark cycles, and regular food and water. Before the experiments, all the mice were given a week to get used to their surroundings, and then 1 × 105 live tumor cells were subcutaneously injected into their left flanks in 200 mL of PBS containing 0.2% BSA (Fig. 2) [12]. After that, mice were separated into eight equal groups at random (Table 1). To assess the immunomodulatory effects of Gardasil, the vaccine was given to one group as a positive control group. All groups received treatment in 100 µl volume and twice a one-week interval. One week after the last agent-therapy sampling was started.

Fig. 2figure 2

Tumor mice model with palpable tumor. A 18 days after injection, B 28 days after injection

Table 1 The characteristics of the studied groupsThe proliferation level of splenocytes

In order to assess the level of splenocytes proliferation, the MTT assay was performed. The splenocytes were placed in 96-well flat-bottomed plates with DMEM media supplemented with 10% FBS (1 × 105 cells/100 μl/well), and they were stimulated with antigens produced from the tumor cells by freezing and thawing (20 μg/ml). The cultures were pulsed with 20 μl of the MTT solution (5 mg/mL) for 4 h at 37 °C after 72 h of incubation. The formazan crystal was then broken down by adding 100 mL of DMSO and vigorously shaking the mixture. Using an ELISA reader, the optical density (OD) at 492 nm was calculated (Dynatech, Denkendorf, Germany). The tests were carried out in sets of three [13].

Lactate dehydrogenase assay

A LDH detection kit was used to examine cytotoxic activities. This assay is a practical, quick colorimetric method for quantifying cytotoxicity by monitoring the activity of LDH produced from injured cells. Most cells contain the cytoplasmic enzyme LDH, which is stable. The TC1 cell line was utilized as the target cells, and the splenocytes served as the effector cells. The assay environment, DMEM with 1% FBS, was used to wash the effector and target cells before they were co-cultured in 96-well round-bottomed plates for six hours at 37 °C at a ratio of 50 effector cells to one target cell. The plates were then centrifuged, and the supernatants were then deposited on 96-well flat-bottomed plates. The LDH detection mixture was then added to each well and placed in the refrigerator at room temperature for 30 min. An ELISA reader from Dynatech, Denkendorf, Germany, was used to measure the OD at 492 nm [13].

Measurement of NO in splenocytes population

Using the Griess reagent to measure the nitrite content of the splenocytes culture supernatants, the potential for NO generation was evaluated. After the splenocytes had been cultivated, 50 ml of the cell-free supernatants were taken and combined with 50 ml of Griess reagent, which contains 0.1% sulfanilamide, 3% phosphoric acid, and 0.1% N-(1-Naphthyl) ethylenediamine. The resulting combination was left to sit at room temperature in the dark for ten minutes. After incubation, an ELISA reader (Dynatech, Denkendorf, Germany) detects the absorbance at 492 nm [13].

Cytokine assay

One week after the last agent-therapy, mice were euthanized to measure the cytokine assay induced in splenocytes. In an aseptic setting, splenocytes were separated from the mice, single-cell suspensions of the splenocytes were made in DMEM medium containing 10% FBS, and RBCs were eliminated using ACK (Ammonium-Chloride-Potassium) Lysing Buffer. After that, 24-well plates were treated with the cell suspensions (2 × 106 cells/mL), and tumor antigens were pulsed into the plates. These antigens were derived from the tumor cells by freezing and thawing (20 μl). As previously mentioned, tumor antigen was created. Following 72 h, the culture supernatants were gathered. According to the manufacturer's instructions, the ELISA kit was used to measure the production of IFN-γ, IL-4, and TGF-β [13].

Statistical analysis

The statistical analysis was conducted by using SPSS Statistics 23 and the Tukey's Test. The results are shown as means ± SD. P < 0.05 was considered statistically significant.

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