The L2RYC cell line was obtained from ATCC (Manassas, VA, USA), and maintained in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO₂. Unless indicated otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Sprague-Dawley (SD) male rats, 4-weeks of age (n=25), were randomly divided into five groups: group 1, plasmid only; group 2, ultrasound + plasmid; group 3, microbubble + plasmid; group 4, ultrasound + microbubbles + plasmid; and group 5, untreated control. The siMDR1-loaded lipid micro-bubble was prepared as described (ref?). Plasmid (50 µg) in 100 µl phosphate-buffered saline (PBS) or lipid microtubule was injected into the tail vein of the experimental rats, and the right testis was exposed to 300 kHz-ultrasound irradiation at the acoustic intensity of 2 W/cm² for 10 min. After 2 weeks, frozen sections of the right testis were observed. Green fluorescent protein (GFP) expression was considered as an indicator of efficiency of gene delivery.

As previously described (14), freshly prepared testis tissues were minced and homogenized in TRIzol reagent and total RNA was extracted by TRIzol method. cDNA templates were generated with 10 µg of total RNA and hexamer using SuperScript II reverse transcriptase (Invitrogen). The PCR primers specific for detecting MDR1 (sense, 5′-GAGAACATCGCCTACGG-3′ and antisense, 5′-GCTTCCTGGACGACCTT-3′); and GAPDH (sense, 5′-TGGATGGTCCCTCTGGAA-3′ and antisense, 5′-GTGAG CTTCCCGTTCAGC-3′) were designed using the Primer 3.0 program. Real-time PCR reactions were carried out using a Bio-Rad protocol as follows: 94°C for 20 sec, 55°C for 20 sec, 70°C for 20 sec for 40 cycles, reading plates after each cycle. Data are reported as the fold-change with endogenous GAPDH normalization.

Western blotting was performed as previously described (14,15). Freshly prepared testis tissues were homogenized in liquid nitrogen and lysed in RIPA buffer with phenylmethanesulfonyl fluoride (PMSF). Approximately 20 µg of total proteins/lane were subjected to 10% SDS-PAGE gel and then electrically transferred to Immobilon-P membranes (Millipore). The membranes were blocked with 5% fat-free skimmed milk in Tris-buffered saline and Tween-20 (TBST) buffer at room temperature for 1 h, followed by incubation with anti-MDR1 or anti-β-actin at 4°C overnight. After washing with TBST, the membranes were probed with the appropriate secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, USA) at room temperature for 1 h. The expression of the proteins was visualized using enhanced chemiluminescent substrate (Kaiji Biotechnology, China) and exposed under the Syngene G:BOX Imaging System.

Daunorubicin accumulation was detected at 2 days after the SD rat treatment. Daunorubicin (2 ml) at a concentration of 0.1 mg/ml was administered via tail vein injection. Testis tissues were collected after 2 h and prepared into frozen sections. Red fluorescence which was emitted by daunorubicin indicated the accumulation/concentration of daunorubicin and was detected under a fluorescence microscope.

Sixty SD male rats 3-weeks and 3-months of age were collected; 10 rats in each group. After being anesthetized by chloral hydrate, the right testis of the rats was exposed by opening the skin, meat membrane and tunica vaginalis. A cell (10 µl) suspension containing 1×10⁶, 1×10⁷ or 1×10⁸ cells was injected into the testis via the connection part of the ductuli efferentes testis (16). Then the testis was put back into the scrotum, the spermatic cord and meat membrane were fixed to prevent retraction, and finally the scrotal incision was sutured. The survival and general situation of the rats were dynamically monitored. The testis volume was detected every 7 days to draw a growth curve. Testis was scanned with Color Doppler Ultrasound Diagnostic instrument to observe the change in testicular size, blood supply and sonographic characteristics. Hematoxylin and eosin (H&E) staining and immunohistochemistry were performed to evaluate tumor progression.

Forty SD male rats 3-weeks of age were collected to set up the testicular tumor model as described above. After 3 weeks, successful constructed testicular tumor-bearing rats were randomly divided into 4 groups: group 1, chemotherapy only; group 2, blank microbubbles + ultrasound + chemo- therapy; group 3, siMDR1 plasmid-loaded microbubbles + ultrasound + chemotherapy; and group 4, saline control. Microbubbles or siMDR1 plasmid-loaded microbubbles were constantly injected into the tail vein and the right testis was exposed to an ultrasound wave field at 300 kHz, 2 W/cm², irradiation for 10 min. At 1 day after microbubble transfection, the rat models were treated with vincristine chemotherapy by intravenous injection 1 time/week, for 2 weeks. Surface area was calculated on the basis of the Meeh-Rubner formula: A (m³) = 9.1 × W (g)^2/3/10,000; rat and human dose conversion formula: trial dose in rats (mg/kg) = dose in human (mg/kg) × 36/6 (human dose conversion factor/rat dose conversion factor). The survival and general situation of rats was dynamically monitored. The volume of the testis was measured every week. Models were sacrificed at 1 week after chemotherapy for H&E staining, immunohistochemistry and Fas/p53 gene detection.

The harvested tumor tissues were fixed with 10% formalin, embedded in paraffin, and serially cut into 5-µm sections. Sections of each specimen were stained with H&E. For immunohistochemistry, the sections were dewaxed in xylene, rehydrated in graded ethanol solutions, and denatured in water at 60°C for 1 h, followed by incubation with α-1-antitrypsin (ATT) antibody at 4°C overnight. After washing in PBS 3 times, the sections were incubated with anti-goat IgG HRP-conjugated secondary antibody at 37°C for 1 h, colored with DAB and counterstained with hematoxylin.

Quantitative data are presented as the mean ± SD. Statistical analysis was performed using the two-tailed Student's t-test or analysis of variance. The probability value of P<0.05 was considered to indicate a statistically significant result.

The MDR1 gene which is highly expressed in the testicular capillary wall, hinders chemotherapy drugs into the testis (4,17). Herein, we aimed to use ultrasound-targeted microbubble destruction to deliver the siMDR1 gene into in vivo target testicular capillaries in vivo, to explore whether the expression and function of the MDR1 gene and P-gp were effectively suppressed.

Since the pSEB-siMDR1 plasmid contains the GFP gene sequence, GFP is an indicator to measure the gene transfection efficiency. As shown in Fig. 1A, GFP expression in testicular interstitial capillary endothelial cells, was only observed in the microbubbles + ultrasound group (group 4), suggesting that the pSEB-siMDR1 plasmid was successfully transfected. There was no GFP expression in the other 4 groups. We further demonstrated that the mRNA expression of the MDR1 gene (Fig. 1B) and protein expression (Fig. 1C) of P-gp were reduced in the microbubble + ultrasound group only. No difference was found between the other 3 intervention and control groups. Daunorubicin as a P-gp substrate, spontaneously emits red fluorescence. At 1 h after daunorubicin injection via tail vein, red fluorescence indicating increased daunorubicin accumulation was observed in the frozen sections of testis in the microbubble + ultrasound group (Fig. 1D), suggesting that the function of P-gp was inhibited and the drug accessed the testis easier. The above results indicated that the combination of ultrasound and microbubbles is an effective method to mediate gene transfection in vivo.

These data suggest that ultrasound microbubble-mediated gene delivery effectively promoted the plasmid DNA transfection in vivo.

Rat yolk sac tumor L2 cells were injected into testicular tissue of the SD rats system to establish the testicular tumor model (Fig. 2A), and then we evaluated the model feasibility and application value by observing the biological characteristics of the tumors, growth rate and morphology. In rats of different ages, stages and different planting cell concentrations, the testicular tumor formation rate and the survival time of the tumor-bearing rats were different (Table I). In the group with implanted cells at the concentration of 1×10⁶/ml, no tumors were present at the end of the experiment. At 3 weeks after implantion of cells at the concentration of 1×10⁷, the tumor formation rate of the 3-week-old rats was 100%, and increased gradually with time after planting, and reached a peak at the 6th week with regions of ulceration and erosion; some rats began to die. The tumor formation rate of the 3-month-old rats was only 12.5%, and tumors increased more slowly. When plantation of the cells was at the concentration of 1×10⁸, the tumor formation rate of both 3-week- and 3-month-old rats were 100%, yet the tumors grew very quickly with a high mortality rate.

The growth curve of testicular tumors (Fig. 2B) showed that the testicular volume of the rats injected with 1×10⁶ cells was similar to that of the control group. With injection of 1×10⁷ tumor cells, the relative volume of the testis was significantly larger than that of the control group, particularly the 3-week-old rats. With injection of 1×10⁸ tumor cells, the testis volume increased rapidly; all rats died after 5 weeks in the 3-week-old rats and 7 weeks in the 3-month-old SD rats, respectively. Thus, we chose to establish the testicular tumor model with the 3-week-old rats and implanted cells at the concentration of 1×10⁷. Ultrasound imaging showed that compared with the control group, the testicular volume of the model side increased significantly; the internal echo was medium and uniform, with small punctuate and cord-like high echo. Part of the testicular tissue exhibited liquefied necrosis with no echo area (Fig. 3A). The affected testis was much larger than the normal side; the testicular mass was suborbicular, cystic and enveloped. Transverse section of the tumor was pale yellow, soft fleshy with partial necrosis (Fig. 3B). Pathological sections under the microscope exhibited loose reticulate structure, regions of adenoid structure, eosinophilic granular and Schiler-Duval bodies by H&E staining. Immunohistochemistry showed that AAT was positively stained (Fig. 3C). Therefore, the testis tumor model was successfully constructed.

This experiment aimed to investigate the treatment efficiency of chemotherapy drugs on testicular tumors following the inhibition of P-gp. Following treatment with chemotherapy drugs, tumor growth was inhibited. The survival rate was improved obviously when compared with the control group at the same time-point. At the end-point, the survival rate of group 3 was increased to 60%, which was significantly different with the other groups (P<0.05). Chemotherapy was reported to inhibit the growth of tumors (18). Relative testis volume was significantly smaller than that of the control group. In our groups, the testicular volume of the ultrasound microbubble-mediated siMDR1 therapy after chemotherapy was the smallest (Fig. 4A and B), indicating the best efficiency of tumor suppression.

H&E staining showed that the numbers of tumor cells within the testicular tissues of group 1–3 were significantly decreased compared with that of group 4, without tumor-specific adenoid structure, and no eosinophilic bodies and Schiler-Duval bodies. Particularly group 3 had visible lumen-like structure in testis tissues. Immunohistochemistry results showed that positive AAT expression of testicular tissues in group 1–3 was lower than that in group 4 (Fig. 5). Therefore, we further confirmed that ultrasound combined with micro-bubble transfection of siMDR1 in the testicular capillary wall provided easier access of vincristine to the testis tissues, and thus improved the effect of chemotherapy on testicular tumors.