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Abstract • Background • Materials and Methods •Results and Discussions •References

Robert W. Bradford D. Sc.1
Kexiang Ding 2
1. Bradford Research Institute, CA, USA
Professor of Capital University, Washington DC
2. FRC Free Radical Biology & Medical Research Center

ABSTRACT

This paper investigated the effect of FRC001 on nonspecific immunity of mice and graft versus host reaction (GVHR). The results shows that FRC001 had some effect on phagocytic function of mononuclear macrophage, spleen index and thymus index, splenic lymphocyte transformation, serum hemolysin formation, host spleen reaction coefficient and stimulate coefficient and stimulus index i.e. FRC001 can increase and enhance humoral immunity and cellular immunity of experimental basis for the use of FRC001 in regulating immunity and preventing and curing tumor.

Key Word : FRC001 Nonspecific immunity graft versus host reaction (GVHR)

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BACKGROUND

Tumor formation is related to immunologic inadequacy in recent year, there are many report on immunologic inadequacy index which was regarded as an important index in the screening of antineoplastic. The study investigated the effect of FRC001 on nonspecific immunity of mice and GVHR.

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MATERIALS AND METHODS

1. Materials

   1.1 Subject : FRC001.
   1.2 Experimental Animal : Mouse, Kunming species, 18~22g, half male and half female, provided by Experimental Animal Center of Jiangsu Tumor Prophtlactico-therapeutic Research Institute.

2. Method

   2.1 Phagocytic function of mononuclear macrophage (mouse carbon clearance test)

   2.1.1 Experimental principle :

   Experimental mouse was injected with Indian ink which was used as granular foreign body. The Indian ink was phagocytized and cleared by mononuclear macrophage after it went into circulation. Ninety percent of it was phagocytized by liver Kupffer cell while the rest was phagocytized by spleen macrophage. The clearance rate of granular foreign body in circulation reflected the phagocytic function of mononuclear macrophage. Within given scope, the clearance rate of granular foreign body had exponential function relation with granular dose, i.e. phagocytosis rate had direct proportion relation with granular concentration in circulation. They have linear relation in system of coordinates with the time as the abscissa and granular concentration as the vertical ordinate. The slope K of the line is the phagocytosis rate (or clearance rate).

   2.1.2 Manipulation.

   Mice were randomly divided into negative control group, positive control group and different dose of FRC001 groups, They were fed with distilled water, elemene milk and different dose of FRC001 for successive 10 days. Each mouse was injected with Indian ink(diluted to 1~5 times) 0.05ml/1/0g.bw from mouse tail vein in 30 minutes after the last feeding. In the first minute (t1) and 5^th minute (t5) after injection 20 m 1 blood was drawn from postobital vein with suction tube in which was moistened with heparin solution and diluted in 2ml 0.1% sodium carbonate solution. Then absorbance (A) of blood solution was assayed at 680nm. To calculate the value of K by following formula. (lgAl-lgA5)/(t5-t1)=(lgA1/A5)/4

   2.2 GVHR test. Two pure line mice C57BL JCR hybridized to give birth to newborn mouse. Beginning at the first day of the birth of the newborn mouse, experimental male parent were fed with FRC001 for 10 days while control male parent were not given any treatment. On the 11^th day after the birth of newborn mouse, male parent mice were killed by dislocation to take out spleens. Spleen cell suspension (1X10 ₈/ml) was prepared by aseptic technique. The newborn mice were divided into experimental group, control group and normal group. The experimental newborn mice were inoculated 0.1 ml spleen cell suspension of experimental male parent, the control newborn mice were inoculated 0.1 ml spleen cell suspension of control male parent, and the normal group were not given any treatment. All these newborn mice were killed at seventh day after inoculation, and their spleens were weighed to calculate spleen coefficient (mg spleen/10g.bw). Then calculation spleen stimulus index (SI) by following formula : SI = (Average spleen coefficient of experimental or control) /average spleen coefficient of normal group.

   2.3 Spleen index and thymus index test

    Kunming species mice (18~22g) were randomly divided into negative control group (fed with N>S) positive control group and FRC001 group. They were fed with respective subject for successive 10 days, and killed by orbital bleeding on the second day after the last feeding. Their spleen and thymuses were stripped out and weighed accurately with torsion balance. The results showed with organ index-weight of spleen or thymus (mg)/10g.bw

   2.4 Mouse lymphocyte transformation test-co;orimetry of mouse splenic MTT

    Pure species Kunming mouse was killed by orbital bleeding and its spleen was stripped out to make 5 X 10 (8) /ml cell suspension with Hank’s solution and RPM 1640 medium cell suspension 100 m 1 was added to pore of culture dish, and every pore was added PHA 100 m 1 and experimental drugs, in the meantime, control pore was set up. They were cultured in 37~38 °C.5% CO2 incubator for 72 hrs. Every pore was added 50 m 1 MTT 4~5 hours before the ending of culture, then continue to culture 24h, 36h, 48h, respectively. They were put into 4 C refrigerator for one night. Supernate 150 m 1 was sucked, added 150 m 1 acid isopropanol and blown to homogeneous solution. Absorbance (A) of every tube was assayed at 630nm of ELISA photometer. To see detail in reference[10].

   2.5 Serum hemolysin test

1.Mice (Kunming species, 18~22g) were randomly divided     into nonimmunologic group, immunologic group immunology and drug (low, middle and high dose) group. Every mouse of the last two groups was injected introper itoneally 0.2 ml sheep red blood cell (SRBC) which diluted to 3:5(V/V) with N.S Mice of drug group were given drug before or after immunization. Four days after immunization. 1 ml blood was taken to get serum. To see the detail in reference [8].

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RESULT AND DISCUSSIONS

1. The effect of FRC001 on phagocytic function of mononuclearmacrophage in mice (Figure 1)

Figure 1. The effect curve of FRC001 on Phagocytic Function of Mononuclearmacrophage in mice.

The increase of carbon clearance index (K) reflects the enhancement of phagocytic function of mononuclearmacrophage and nonspecific immunity. According to Figure 1, low, middle and high dose of FRC001 increased K value significantly (p<0.01). The result showed FRC001 can enhance the phagocytic function of mononuclearmacrophage and nonspecific immunity.

2. The effect of FRC001 on spleen index and thymus index. (Table 1)